Systems and methods for growth of intestinal cells in microfluidic devices

ABSTRACT

Organs-on-chips are microfluidic devices for culturing living cells in micrometer sized chambers in order to model physiological functions of tissues and organs. Engineered patterning and continuous fluid flow in these devices has allowed culturing of intestinal cells bearing physiologically relevant features and sustained exposure to bacteria while maintaining cellular viability, thereby allowing study of inflammatory bowl diseases. However, existing intestinal cells do not possess all physiologically relevant subtypes, do not possess the repertoire of genetic variations, or allow for study of other important cellular actors such as immune cells. Use of iPSC-derived epithelium, including IBD patient-specific cells, allows for superior disease modeling by capturing the multi-faceted nature of the disease.

CROSS-REFERENCED TO RELATED APPLICATIONS

This application is a continuation of and claims priority to, co-pendingU.S. Non-Provisional application Ser. No. 16/051,004, filed Jul. 31,2018, and National Entry of PCT Application Serial NumberPCT/US17/16079, filed Feb. 1, 2017, and Provisional Application,62/437,314, filed Dec. 21, 2016, now expired; Provisional Application,62/354,040, filed Jun. 23, 2016,now expired; Provisional Application,62/332,849, filed May 6, 2016, now expired; and Provisional Application,62/289,521, filed Feb. 1, 2016, all now expired, each of which is hereinincorporated by reference in its entirety.

SEQUENCE

A Sequence Listing has been submitted in an ASCII text file named“18530_ST25.txt” created on Apr. 20, 2022, consisting of 7,030 bytes,the entire content of which is herein incorporated by reference,

FIELD OF THE INVENTION

The present invention relates to a combination of cell culture systemsand microfluidic fluidic systems. More specifically, in one embodiment,the invention relates microfluidic chips seeded with stem-cell-derivedcells that mature and.or differentiate into intestinal cells. In oneembodiment, the stems are induced pluripotent stem cells (hiPSCs) andthe intestinal cells are foregut cells. In some emdodiments, such forgutchips are tested for effects of endocrine disrupting chemicals (EDCs)during critical periods in tissue development mimicking critical periodsof fetal development for short and long term downstream effects. Inparticular, methods for use are provided for induced pluripotent stemcells (hiPSCs) to elucidate adverse effects and mechanisms of chroniclow-dose EDC exposures on developing gut and hypothalamicneuropeptidergic neurons, and serves as a platform for mimicking the inutero exposure to EDCs. Morover, in yet further embodiments, iPS cellsderived from obese individuals are seeded on chips for determing effectsof EDCs in relation to obsesigens.

The invention further relates to methods and systems for providing cellsfrom intestinal organoids (the organoids derived from iPSCs) onmicrofluidic chips. In one embodiment, additional cells are on the chip,e.g. induced neuronal cells. In some embodiments, microfluidicintestinal Organ-On-Chips mimic human gastrointestinal disorders, e.g.IBD, etc.

BACKGROUND

Persistent human exposure to elevated levels of man-made endocrinedisrupting chemicals (EDCs) during critical periods in fetal developmentmay lead to long-term disruption of metabolic homeostasis in endocrinetissue progenitors, thus contributing to childhood obesity.Specifically, endocrine control of feeding behavior involves theparticipation and communication between the hypothalamic arcuate nucleusand the gastrointestinal tract enteroendocrine cells, stomach inparticular. The hypothalamic (HT) neuropeptidergic neurons receiveendocrine signals from parts of gut including gastrin and ghrelin fromstomach, peptide YY from intestine and bring about orexigenic oranorexigenic effects. Hence, abnormalities during development due toexternal or environmental factors such as EDCs may play a role indysfunction of the gut-brain interactions thereby bringing about feedingdisorders and obesity.

There is paucity of data on the developmental effects of early exposureof EDCs on dysfunction of cells involved in feeding and hunger largelydue to the implausibility of accessing human fetal tissue at differentdevelopmental stages. To fill this void, the Inventors employed humaninduced pluripotent stem cells (hiPSCs) to elucidate the adverse effectsand mechanisms of chronic low-dose EDC exposures on developing gut andhypothalamic neuropeptidergic neurons, and serves as a platform formimicking the in utero exposure to EDCs. This is the first suchapplication of the pluripotent stem cell technology.

Without affecting cell viability, low-dose EDCs significantly perturbedNF-κB signaling in endocrinally active iFGEs and iHTNs. Consequently,EDC treatment decreased maximal mitochondrial respiration and sparerespiratory capacity in iFGEs and iHTNs upon mitochondrial stresschallenges, likely via NF-κB mediated regulation of mitochondrialrespiration and decreased expression of both nuclear (SCO2, TFAM,POLRMT) and mitochondrially-encoded (CytB5) respiratory genes. Treatmentwith NF-κB inhibitor, SN50, rescued EDC-induced aberrant NF-κB signalingand improved mitochondrial respiration. This seminal work is the firstreport about a human pluripotent stem cell (PSC)-based mechanistic modelof endocrine disruption by environmental chemicals, describing theadverse impact of EDCs on NF-κB signaling and mitochondrial dysfunction.This paves the way for a reliable screening platform for obesogenic EDCsin the developing human endocrine system.

SUMMARY OF THE INVENTION

The invention provides a method of compound screening, comprising:providing a quantity of differentiated induced pluripotent stem cells(iPSCs); contacting the differentiated iPSCs with one or more compounds;measuring one or more properties of the differentiated iPSCs, whereinmeasurement of the one or more properties of the differentiated iPSCsidentifies characteristics of the one or more compounds. In oneembodiment, said compound screening comprises screening for endocrinedisruption. In one embodiment, said characteristics of the one or morecompounds comprise inducing phorphorylation of Nuclear factor kappa B(NF-kB). In one embodiment, said characteristics of the one or compoundscomprise decrease in mitochondrial respiration. In one embodiment, saidcharacteristics of the one or compounds comprise decrease in expressionof one or more of Cytochrome C Oxidase Assembly Protein (SCO2), RNAPolymerase Mitochondrial (POLRMT),Transcription Factor A, Mitochondrial(TFAM) and CYTB5. In one embodiment, said differentiated iPSCs areforegut epithelium. In one embodiment, said differentiated iPSCs arehypothalamic neurons.

The invention provides a method of differentiating induced pluripotentstem cells, comprising: providing a quantity of induced pluripotent stemcells (iPSCs); and culturing in the presence of one or more factors,wherein the one or more factors are capable of differentiating theiPSCs. In one embodiment, said iPSCs are differentiated into definitiveendoderm by culturing in the presence of one or more factors comprisingActivin A and Wnt3A. In one embodiment, said culturing in the presenceof one or more factors comprising Activin A and Wnt3A is for about 3days. In one embodiment, said differentiated iPSCs are initiallycultured under serum-free conditions, followed by addition of serum. Inone embodiment, said definitive endoderm is differentiated into foregutspheroids by further culturing in the presence of one or more factorscomprising CHIR99021, FGF (FGF4), LDN (small molecule), and RetinoicAcid (RA). In one embodiment, said culturing in the presence of one ormore factors comprising CHIR99021, FGF (FGF4), LDN, and Retinoic Acid(RA) is for about 3 days. In one embodiment, said foregut spheroid isdifferentiated into foregut epithelium by culturing on a coated surface.In one embodiment, said foregut spheroid is differentiated into foregutepithelium by additional culturing in the presence of one or morefactors comprising epidermal growth factor (EGF). In one embodiment,said additional culturing in the presence of one or more factorscomprising epidermal growth factor (EGF) is for about 20 days. In oneembodiment, said iPSCs are initially cultured in the presence ofROCK-inhibitor Y27632. In one embodiment, said iPSCs are differentiatedinto neuroectoderm by culturing in the presence of one or more factorscomprising LDN193189 and SB431542. In one embodiment, said culturing inthe presence of one or more factors comprising LDN193189 and SB431542 isfor about 2 days. In one embodiment, said neuroectoderm isdifferentiated into ventral diencephalon by culturing in the presence ofone or more factors comprising smoothened agonist SAG, purmorphamine(PMN) and IWR-endo. In one embodiment, said culturing in the presence ofone or more factors comprising moothened agonist SAG, purmorphamine(PMN) and IWR-endo is for about 5-6 days. In one embodiment, saidventral diencephalon is matured by culturing in the presence of one ormore factors comprising DAPT, retinoic acid (RA). In one embodiment,said culturing in the presence of one or more factors comprising DAPT,retinoic acid (RA) is for about 4-5 days. In one embodiment, said matureventral diencephalon is further matured by culturing in the presence ofone or more factors comprising BDNF. In one embodiment, said culturingin the presence of one or more factors comprising BDNF is for about20-27 days.

Endocrine disrupting chemicals (EDCs) are contemplated to affect earlytissue development either by causing immediate damage or causing analteration considered harmful to an organism, such as an immediatechange to one or more of a cell function, tissue function, physiologicalfunction, developmental pathway; and/or by causing damage over longerterm in a subtle or unexpected way, i.e. as deleterious during earlytissue development. Example 19 discusses some of these tissue changes.

We hypothesized that chronic low-dose exposure to endocrine disruptingchemicals (EDCs), is deleterious during early human endocrine tissuedevelopment. Further, we hypothesized that such exposure results inhyperactive NF-κB and HMG protein pro-inflammatory signaling withpermanent mitochondrial dysfunction.

Inflammatory bowel disease (IBD), such as Crohn's disease and ulcerativecolitis, involve chronic inflammation of human intestine. Mucosal injuryand villus destruction are hallmarks of IBD believed to be caused bycomplex interactions between gut microbiome, intestinal mucosa, andimmune components. It has been difficult to study the relativecontributions of these different factors in human intestinalinflammatory diseases, due to a lack of animal or in vitro modelsallowing for independent control of these parameters. As a result,existing models of human intestinal inflammatory diseases rely either onculturing an intestinal epithelial cell monolayer in static culture ormaintaining intact explanted human intestinal mucosa ex vivo. Given thedynamic tissue environment of the gut, these static in vitro methodscannot faithfully recapitulate the pathophysiology of human IBD.Notably, intestinal epithelial cells cultured in plates completely failto undergo villus differentiation, produce mucus, or form the variousspecialized cell types of normal intestine.

Organs-on-chips are microfluidic devices for culturing living cells inmicrometer sized chambers in order to model physiological functions oftissues and organs. Continuous perfusion through the chambers allowsincorporation of physical forces, including physiologically relevantlevels of fluid shear stress, strain and compression, for study oforgan-specific responses. Of great interest is adapting such fabricationtechniques for development of a “gut-on-a-chip” capable of replicatingthe corresponding physiological environment, and dynamicallyincorporating those multiple components (microbiome, mucosa, immunecomponents) in a manner mirroring IBD pathophysiology. Towards theseaims, prior attempts have successfully relied on human intestinalepithelial cells (Caco-2) cultured in the presence of physiologicallyrelevant luminal flow and peristalsis-like mechanical deformations. Thisapproach allows formation of intestinal villi lined by all fourepithelial cell lineages of the small intestine (absorptive, goblet,enteroendocrine, and Paneth), with enhanced barrier function,drug-metabolizing cytochrome P450 activity, and apical mucus secretion.

However, a chief limitation of existing approaches is that carcinomalines such as Caco-2 cells do not possess the intestinal epithelialsubtypes. As such, the impact of bacteria and/or inflammatory cytokineson various intestinal subtypes cannot be determined. Additionally,Caco-2 cells do not possess the repertoire of genetic variations nowunderstood to be associated with IBD, thereby limiting opportunity tofurther evaluate response of IBD genetic factors. Finally, existingmodels fail to incorporate other cell types, such as immune cells (e.g.,macrophages, neutrophils, and dendritic cells) to investigate their rolein disease pathology. Thus, there is a great need in the art toestablish improved gut organ chip models that faithfully incorporatethese multi-faceted elements.

To test this, the gastrointestinal organoids (iGIOs) and hypothalamicneurons (iHTNs) seeded on “organ-on-chip” microfluidic device areexposed to chronic low-dose treatments (TDI range) of EDCpollutants/mixtures.

As an example, in some embodiments, iPSC lines derived from obeseindividuals were used in testing on microfluidic chips for responses tocompounds, including but not limited to endocrine disrupting chemicals(EDCs), i.e. obesogens, e.g. as chronic low-dose treatments (TDI range)of EDC pollutants/mixtures (e.g. tributyltin (TBT), perfluorooctanoicacid (PFOA), butylated hydroxytoluene (BHT), and bis(2-ethylhexyl)phthalate (DEHP), etc. Testing is contemplated to include determiningsigns of detrimental effects of exposure to putative endocrinedisrupting chemicals in developing cells i.e. iHTNs and iFGEs, with anexample of analysis including but not limited to dysregulated secretedprotein groups will be identified by quantitative proteomics. Exemplaryresults are described in Example 32.

The invention provides a method of manufacturing a microfluidicapparatus comprising a population of intestinal cells with an organizedstructure, comprising: disaggregating human intestinal organoids (HIOs)into single cells; and adding the single cells to the apparatus. In oneembodiment, said single cells are purified based on CD326+ expressionbefore addition to the apparatus. In one embodiment, said adding thesingle cells to the apparatus comprises resuspension in a mediacomprising one or more of: ROCK inhibitor, SB202190 and A83-01. In oneembodiment, said human intestinal organoids (HIOs) are cultured in thepresence of ROCK inhibitor prior to disaggregation. In one embodiment,said human intestinal organoids (HIOs) are derived from inducedpluripotent stem cells (iPSCs). In one embodiment, said iPSCs arereprogrammed lymphoblastoid B-cell derived induced pluripotent stemcells (LCL-iPSCs). In one embodiment, said iPSCs are reprogrammed cellsobtained from a subject afflicted with an inflammatory bowel diseaseand/or condition.

The invention provides a method of manufacturing a microfluidicapparatus comprising a population of intestinal cells with an organizedstructure, comprising: disaggregating human intestinal organoids (HIOs)into single cells; and adding the single cells to the apparatus. In oneembodiment, said single cells are purified based on CD326+ expressionbefore addition to the apparatus. In one embodiment, said adding thesingle cells to the apparatus comprises resuspension in a mediacomprising one or more of: ROCK inhibitor, SB202190 and A83-01. In oneembodiment, said human intestinal organoids (HIOs) are cultured in thepresence of ROCK inhibitor prior to disaggregation. In one embodiment,said human intestinal organoids (HIOs) are derived from inducedpluripotent stem cells (iPSCs). In one embodiment, said derivation ofhuman intestinal organoids (HIOs) from induced pluripotent stem cells(iPSCs) comprises: generation of definitive endoderm by culturinginduced pluripotent stem cells (iPSCs) in the presence of Activin A andWnt Family Member 3A (Wnt3A); differentiation into hindgut by culturingdefinitive endoderm in the presence of FGF and either Wnt3A orCHIR99021; collection of epithelial spheres or epithelial tubes;suspension of epithelial spheres or epithelial tubes in Matrigel; andculturing in the presence of CHIR99021, noggin and EGF. In oneembodiment, said apparatus comprises an organized structure comprisingvilli. In one embodiment, said villi are lined by one or more epithelialcell lineages selected from the group consisting of: absorptive, goblet,enteroendocrine, and Paneth cells. In one embodiment, said organizedstructure possesses barrier function, cytochrome P450 activity, and/orapical mucus secretion.

The invention provides a microfluidic apparatus comprising: a populationof intestinal cells, wherein the population comprises an organizedstructure. In one embodiment, said organized structure comprises villi.In one embodiment, said villi are lined by one or more epithelial celllineages selected from the group consisting of: absorptive, goblet,enteroendocrine, and Paneth cells. In one embodiment, said organizedstructure possesses barrier function, cytochrome P450 activity, and/orapical mucus secretion. In one embodiment, said intestinal cells arederived from human intestinal organoids (HIOs) disaggregated into singlecells and purified based on CD326+ expression. In one embodiment, saidhuman intestinal organoids (HIOs) are derived from iPSCs by a methodcomprising: generation of definitive endoderm by culturing iPSCs in thepresence of Activin A and Wnt3A; differentiation into hindgut byculturing definitive endoderm in the presence of FGF and either Wnt3A orCHIR99021; collection of epithelial spheres or epithelial tubes;suspension of epithelial spheres or epithelial tubes in Matrigel; andculturing in the presence of CHIR99021, noggin and EGF.

The use of microfluidic intestinal chips described hereinimproves/increases maturation of iPS derived intestinal cells. Morespecifically, use of such chips improves maturation efficiency, e.g. iPScell differentiation into foregut increases numbers of cells such assynaptophysin (SYP) positive cells, and improves quality of intestinalepithelium, i.e. an epithelial layer folds into finger-like projectionslined with epithelial cells of which some are separated by pit-likeareas mimicking villus-like structures lined with epithelium andpit-like areas, for mimicking human intestinal microvillus when seededwith iPSC derived intestinal cells. Further, these villus structures arecontinuously growing as basal cells divide and move up the sides of thevilli. Moreover, the folds of epithelium comprise non-epithelialintestinal cells.

Moreover, the chip provides an environment where a “complete” set ofrelevant non-epithelial cell types can develop. These non-epithelialintestinal cells include but are not limited to goblet cells, Panethcells, endocrine cells, etc.

The invention provides On-chip differentiation/maturation of cells andtissues, including but not limited to intestinal tissue, epithelium.During the development of the present inventions, the inventorsdiscovered that a flow condition promotes the maturation and/ordifferentiation of intestinal cells forming finger-like/villi-likeprojections. Further, it was discovered that flow of media promotes theformation of tight cell-to-cell junctions, which in some embodimentsthese tight cell-to-cell junctions are detected by TEER measurements,and/or cell-to-cell junctions are detected by cell permeability assays.

One restriction on the use of intestinal enteroids (and cells) derivedfrom human iPS cell lines is that these cells need to be used during acertain time period for producing viable and reproducible microfluidicintestinal chips. However, during the development of the presentinventions, methods and conditions were developed for using multiplealiquots (i.e. duplicate samples) of the same human intestinal enteroidcells in experiments separated by long time periods from the firstexperiment using these cells. Alternatively, intestinal enteroid cellsderived from human iPS cell lines may be stored long term before use ina microfluidic chip.

As shown herein, the inventors discovered that human intestinal Caco-2cell lines as representative intestinal epithelial cells grown in chipswere found to show responses to compounds that were significantlydifferent when compared to responses of intestinal epithelial grown onmicrofluidic intestinal organ-on-chips. Therefore, use of stem cellderived intestinal cells in these chips are improvements over the use ofCaco cells (e.g. the stem cell derived cells have a proper response tointerferon gamma, cellular production of antimicrobials). In particular,the wide range of Caco-2 cell lines used over the last twenty years aresubpopulations and/or clones of cells that were originally obtained froma human colon adenocarcinoma. In part because of their capability tospontaneously differentiate to form monolayers having similarcharacteristics to enterocyte/epithelial layers, Caco-2 cell lines areextensively used as a model of the intestinal barrier and intestinalepithelial cell function. However, during development of the presentinventions microfluidic intestinal chips showed responses to compoundsthat are more similar to human intestinal epithelial responses,considered “proper” responses, than Caco-2 cell lines (e.g. properresponses to interferon gamma, cellular production of antimicrobials,etc.). Therefore, the use of microfluidic intestinal organ-on-chipsdescribed herein, are an improvement over using Caco-2 cell lines.Moreover, primary intestinal cells also show a more natural phenotypethan Caco2 cells when growing on microfluidic chips.

The use of microfluidic intestinal chips described herein show thatdiseases may be modeled using microfluidic chips described herein. Inparticular, microfluidic chips comprising iPSC derived intestinal cells,are contemplated for use as disease models, in particular for intestinaldiseases such as gastrointestinal disorders, inflammatory intestinaldisorders, gastrointestinal cancer cells, gastrointestinal cancerdevelopment, gastrointestinal tumors, polyps, cells derived fromgastrointestinal tissue, etc. In some embodiments, cells for use inproducing iPS cells may be obtained from patients having a range ofInflammatory bowel diseases (IBD) involving chronic inflammation of asmall patch in the digestive tract up to large regions, e.g. colitis,ulcerative colitis, Crohn's disease, etc. Thus, white blood cells fromIBD patients may be used for producing iPS cells for personalized chips.For comparisons, white blood cells from IBD patients may be used forproducing iPS cells. In some embodiments, cell components, microbialcomponents, etc. may be directly obtained from a healthy person, apatient showing symptoms of and IBD, fluid samples and biopsies.

The use of microfluidic chips and systems described herein, apersonalized therapy can be tested in the chip before being used in thepatient. It is well known in the field that not every patient diagnosedwith the same disease responds in the same manner to a treatment. Thus,testing a therapy in the chip using the cells of the very same personthat will be treated, allows determination (e.g. prediction) of how thatpatient will respond. Similarly, diagnostic tests can be done in orderto identify the nature of the disease and then determine a propertherapy, e.g. for reducing or eliminating symptoms, or curing thedisorder or the disease.

Microfluidic intestinal chips described herein are contemplated for usein personalized medicine (e.g. individual patient derived) fordeveloping treatments, including but not limited to disorders, diseasesand cancer, (e.g. individual patient derived). Such use includes but notlimited in use in personalized components i.e. iPS-derived cell typessuch as immune cells or bacteria from stool samples.

Further, personalized chips may be used for tissue analysis, e.g.capability to develop normal intestinal structures and cells from iPCs,responses of iPSC derived intestinal cells to compound testing, e.g.cytokines, drug testing, treatment, etc. Such chips are not limited toone type of patient derived cell and are contemplated for use in growingpersonalized chips with other personalized components, including but notlimited to a particular iPS-derived cell type for use in derivingintestinal cells, such as white blood cells; and other types of cellsthat are contemplated for use in microfluidic intestinal chips, such asimmune cells, including resident, e.g. obtained or derived from tissuebiopsies, cell collection from fluids, isolated from tumors, obtainedfrom populations of circulating white blood cells from patient bloodsamples, genetically modified patient's cells for testing responses ortesting for use in treatments; or other types of patient samples, suchas microorganisms, e.g. bacteria, fungi, viruses, isolated from stoolsamples that may be added to the patients iPSC derived intestinal cellson a personalized microfluidic organ-on-chip. In fact, an individualizeintestinal chip may further comprise biological components for testingthat are not derived from the patient, such as microorganisms,genetically modified cells, including microorganisms, for use in testingtreatments.

While personalization was discussed above, the personalized therapydeveloped for one patient, can be used to treat another patient. As oneexample, the treatment developed for one patient may then be used totreat another patient, e.g. a patient considered having a similargenetic match, such as an identical twin, sibling, parent, grandparent,relative, etc.

Microfluidic intestinal organ-on-chips described herein are contemplatedfor use in isogenic experiments where a cell or tissue is altered (e.g.express a new gene and/or protein, remove a gene or protein, e.g. reduceexpression of that gene or protein) and then compare that altered cellor tissue with a control cell or tissue of the same genotype orphenotype that is not altered.

Isogenic cell lines refer to a population of cells that are selected orengineered to model the genetics of a specific patient population, invitro. Isogenic human disease models include isogenic cell lines thathave genetically matched ‘normal cells’ to provide an isogenic systemfor use in researching disease biology and testing therapeutic agents.

Thus in one embodiment, iPSCs of matching genetics, i.e. clones, areseparated into at least two samples, wherein one sample is used for acontrol, compared to one or more of the samples that is geneticallyengineered to alter expression of one or more genes of interest, e.g.increase gene expression by overexpressing gene(s), i.e. by usingtransient or constitutive expression vectors, knock-in gene expression,specific or nonspecific; or lower the amount of gene expressed, as isunderexpressed, i.e. by using silencing constructs or gene knock-outs(in transient or constitutive expression vectors); or gene editing, i.e.clustered regularly interspaced short palindromic repeats (CRISPR)mediated gene editing, etc. However, it is not intended to limit how anisogenic experiment is done, with nonlimiting examples provided herein,so long as there is a matched control.

Thus in one embodiment, a gene of interest in inserted into the genomeof an iPS cell or derived organoid cell, for comparison to duplicatesamples of cells that are not modified by this insertion. In someembodiments, instead of changing expression levels, a gene is mutated ina cell for comparison to duplicate cell samples not having thatmutation. In some embodiments, cells are altered or mutated prior toseeding microfluidic chips. In other embodiments, cells are altered ormutated after seeding into microfluidic chips. In some embodiments,instead of altering a gene, an expressed protein from DNA inserted intothe genome of a cell is altered, e.g. such as for gene therapy. In someembodiments, an expression DNA vector or RNA for expressing a protein isintroduced into the cell, e.g. such as for gene therapy.

In one embodiment, sources of iPSC derived intestinal cells containingan endogenous mutation in one or more genes of interest are selected foruse in deriving intestinal cells for seeding organ-on-chips. Forcomparison, e.g. control, matching sources of iPSCs may be selected withsimilar or the same genetic background that do not have the samemutations in the one or more genes of interest.

Microfluidic intestinal organ-on-chips described herein are contemplatedfor use in modeling obesity related disorders including but not limitedto obese individuals without additional symptoms and obese individualsfurther showing symptoms including prediabetic, diabetic, i.e. Type Iand Type II diabetes, etc. For example, during the development of thepresent inventions, iPSC lines were generated from individuals withnormal body mass index (BMI<25) and individuals considered super obese(SO) with BMI>50, then tested on-chip. These obese iPSC werere-differentiation into endocrine tissues-gastrointestinal (GI)organoids and hypothalamic (HT) neuropeptidergic neurons. Thus,Gastrointestinal organoids (iGIOs) and hypothalamic neurons (iHTNs) wereused for seeding into obese modeling microfluidic chips. See. Example31. Differential baseline whole cell proteome profiles were generatedfor these individuals from their iPSC-endocrine cells. Differentiationof iPSCs to gastrointestinal organoids (iGIOs) and hypothalamic neurons(iHTNs) was done in advance of seeding cells on “organ-on-chip”microfluidic devices.

As described herein, microfluidic organ-on-chips comprise neurons alongwith intestinal cells on the same chip. Such neurons include bothiPS-derived and not, (e.g. primary cells) but are not limited to thesetypes of nerves. Thus, in some embodiments, primary neuronal cells, suchas isolated from biopsies, may be added to chips. In some embodiments,neuronal cells may be grown in culture for adding to chips. Further,observation and analysis of chips seeded with iHNs showed thespontaneous development of a lumen area in the lower channel surroundedby neuronal cells.

As described herein, selecting proper cells before seeding on the chipprovides chips mimicking intestinal epithelium (lining) havingvilli-like structures and a range of non-epithelial intestinal cells.During the development of the present inventions it was discovered thatdisassociation of enteroids into single cell suspensions then sortingcells using E-cadherin selection markers for seeding E-cadherin+ cellsinto the apical channel of chips, provided intestinal cell layers havingfinger-like projections and mimicking folding of in vivo intestinalepithelial layers with villus structures. Further, it was discoveredthat the use of a selection reagent for lifting cells from organoidcultures provided single cell suspensions for seeding onto chipsproviding equal or better quality epithelium. Thus, the use of aselection reagent can replace the cell-sorting step.

The present invention, in one embodiment, contemplates a method ofculturing cells, comprising: a) providing a fluidic device comprising amembrane, said membrane comprising a top surface and a bottom surface;b) seeding iPS-derived cells on said top or bottom surface; and c)culturing said seeded cells under conditions that support the maturationand/or differentiation of said seeded cells into intestinal cells. Inone embodiment, said intestinal cells are selected from the groupconsisting of foregut intestinal epithelial cells, midgut intestinalepithelial cells and hindgut intestinal epithelial cells. In oneembodiment, the seeded cells differentiate into Paneth cells, endocrinecells and/or goblet cells. In a preferred embodiment, the seeded cellsare cultured under flow conditions. It is not intended that the presentinvention be limited by the precise configuration of the device or theposition of the cells. In one embodiment, the iPS-derived cells areseeded on said top surface and said method further comprises seedingcells of a second type on said bottom surface. A variety of readouts iscontemplated to assess the cells. In one embodiment, said intestinalcells exhibit a more mature electrophysiology as compared to the sameintestinal cells cultured in a static culture. In one embodiment, theculture under flow conditions results in the formation of villi. In oneembodiment, the seeded cells are (before seeding) selected out from thetotal population of cells to ensure that intestinal cells and/or theirprecursors are favored for seeding. To achieve this, the seeded cellsare, in one embodiment, derived, selected or extracted from organoids.In one embodiment, the selected cells comprise foregut progenitors,midgut progenitors and/or hindgut progenitors. While a variety ofmammalian sources of organoids are contemplated, in a preferredembodiment, said organoids are derived from human induced pluripotentstem cells. It is not intended that the present invention be limited bythe selection method, extraction method or derivation method. In oneembodiment, a biomarker is used to identify the appropriate precursor.In one embodiment, said seeded cells are selected from said organoidusing a selection reagent. The present invention contemplates that thecells can be used to model disease. In one embodiment, said organoidsare derived from induced pluripotent stem cells from a human patientdiagnosed with a gastrointestinal disorder. In one embodiment, saidinduced pluripotent stem cells are from a patient diagnosed withInflammatory bowel disease (IBD). In one embodiment, said inducedpluripotent stem cells are from a patient diagnosed with colitis. Flowcan promote maturation and differentiation of the intestinal cells. Inone embodiment, flow conditions comprise flowing culture media at a flowrate so as to create a shear force. In one embodiment, said flowpromotes the formation of tight cell-to-cell junctions. In oneembodiment, the method further comprises detecting said tightcell-to-cell junctions. This can be done in a number of ways. In oneembodiment, said tight cell-to-cell junctions are detected by TEERmeasurements. In one embodiment, said tight cell-to-cell junctions aredetected by cell permeability assays.

As noted above, the device can be configured in a number of ways. In oneembodiment, said top surface of said membrane defines the bottom surfaceof a first channel and wherein said bottom surface of said membranedefines a top surface of a second channel. It is not intended that thepresent invention be limited to just the use of intestinal cells; othercells and agents can be employed together with the intestinal cells. Inone embodiment, the method further comprises bringing immune cells,cytokines and/or microorganisms (e.g. bacteria, fungi, viruses) intocontact with said intestinal cells. In one embodiment, bacteria arebrought into contact with said intestinal cells. Bringing the bacteria(whether pathogenic or normal flora) into contact with the intestinalcells allows for study of the interaction of these cells. In addition,it allows for drug testing. In one embodiment, the method furthercomprises testing candidate antimicrobials against said bacteria.Bringing a virus into contact with the intestinal cells allows for studyof the interaction of a virus with these cells. In addition, it allowsfor drug testing. In one embodiment, the method further comprisestesting candidate antivirals.

The present invention contemplates that the intestinal cells expressappropriate markers. In one embodiment, said intestinal cells expressthe marker E-Cadherin. The present invention also contemplates that theintestinal cells secrete molecules (e.g. cytokines, antimicrobials,etc.). In one embodiment, the method further comprises the step ofdetecting the production of antimicrobials (or cytokines) by saidintestinal cells.

The present invention contemplates a variety of protocols for culturingthe cells. It is not intended that the present invention be limited toany particular culture time period. In one embodiment, said culturing ofstep c) is performed for at least four days, more typically seven days,or ten days, or even 14 days, or more.

The present invention contemplates introducing factors into the culturemedia to enhance maturation and differentiation. In one embodiment, saidculture media comprises one or more growth factors (e.g. Noggin, EGF,etc.).

The fluidic device can have a number of features. In one embodiment,said fluidic device further comprises at least one inlet port and atleast one outlet port, and said culture media enters said inlet port andexits said outlet port.

The present invention also contemplates, in one embodiment, a method ofculturing cells, comprising: a) providing a microfluidic devicecomprising a membrane, said membrane comprising a top surface and abottom surface; b) seeding stem-cell derived organoid cells on said topsurface so as to create seeded cells; c) exposing said seeded cells to aflow of culture media for a period of time; and d) culturing said seededcells under conditions such that organoid cells mature and/ordifferentiate into intestinal cells. “Intestinal cells” can be of anumber of types. In one embodiment, said intestinal cells intestinalcells are selected from the group consisting of foregut intestinalepithelial cells, midgut intestinal epithelial cells and hindgutintestinal epithelial cells. The microfluidic device can have a numberof designs/configurations (e.g. one channel, two channels, threechannels or more). In one embodiment, said microfluidic device comprisesa first microfluidic channel in fluidic communication with said topsurface of said membrane and a second microfluidic channel in fluidiccommunication with said bottom surface of said membrane, said first andsecond microfluidic channels each comprising a surface that is parallelto said membrane, and each comprising side walls. It is not intendedthat the present invention be limited to just one type of cell in themicrofluidic device; other cell types (in addition to intestinal cells)can be employed. In one embodiment, hypothalamic neurons are in saidsecond microfluidic channel. While not limited to any particularposition for these cells, in one embodiment, hypothalamic neurons growon the parallel surface and side walls of the second microfluidicchannel so as to form a lumen. Again, it is desired that the intestinalcells (or their precursors) express the appropriate biomarkers. In oneembodiment, said intestinal cells (or their precursors) express themarker E-Cadherin.

While the cells are cultured within the microfluidic device, the presentinvention contemplates that they can be assessed either by transparentwindows, by taking the device apart, by collecting cells (or cellproducts) from the outlet ports, or even by sectioning (cutting,slicing, etc.) through a portion of the device. In a preferredembodiment, the method further comprises the step of sectioning saidfirst or second channel and visualizing said cells (with or withoutstaining the cells, with or without reacting the cells with antibodies,etc.).

The present invention contemplates a variety of protocols for culturingthe cells. It is not intended that the present invention be limited toany particular culture time period. In one embodiment, said culturing ofstep c) is performed for at least four days, more typically seven days,or ten days, or even 14 days, or more.

As noted above, the microfluidic device can have a number of designs andfeatures. In one embodiment, said microfluidic device further comprisesat least one inlet port and at least one outlet port, and said culturemedia enters said inlet port and exits said outlet port.

While the organoids can be put into the microfluidic device, it ispreferred that the cells are first separated from the organoids intosingle cells. Moreover, it is preferred that the desired cells areselected, sorted (e.g. using FACS), extracted or otherwise derived fromthe organoid. In one embodiment, said organoid cells were selected orextracted from organoids and comprise foregut progenitors, midgutprogenitors and/or hindgut progenitors. In one embodiment, saidorganoids are derived from human induced pluripotent stem cells. In oneembodiment, said seeded cells were selected from said organoid using aselection reagent. In one embodiment, said seeded cells, after beingselected using a selection reagent, were frozen, stored and subsequentlythawed prior to step b). Storage can be for days, weeks, months or more.

The microfluidic device can be used to study disease. In one embodiment,said organoids are derived from induced pluripotent stem cells from ahuman patient diagnosed with a gastrointestinal disorder. While notintending to be limited to any particular disorder, in one embodiment,said induced pluripotent stem cells are from a patient diagnosed withInflammatory bowel disease (IBD). In another embodiment, said inducedpluripotent stem cells are from a patient diagnosed with colitis.

A variety of culture conditions are contemplated. In one embodiment,said culture media comprises one or more growth factors (Noggin, EGF,etc.).

In an alternative embodiment, the present invention contemplates amethod of culturing cells, comprising: a) providing i) stem-cell derivedorganoid cells and ii) a microfluidic device comprising a membrane, saidmembrane comprising a top surface and a bottom surface; b) subjectingsaid organoid cells to a selection reagent to generate selected cells;c) freezing and storing said selected cells; d) thawing and seeding saidselected cells on said top surface of the membrane of said microfluidicdevice so as to create seeded cells; e) exposing said seeded cells to aflow of culture media for a period of time; and f) culturing said seededcells under conditions such that said selected cells mature and/ordifferentiate into intestinal cells. In one embodiment, said intestinalcells intestinal cells are selected from the group consisting of foregutintestinal epithelial cells, midgut intestinal epithelial cells andhindgut intestinal epithelial cells. While a variety ofdesigns/configurations are contemplated, in one embodiment, saidmicrofluidic device comprises a first microfluidic channel in fluidiccommunication with said top surface of said membrane and a secondmicrofluidic channel in fluidic communication with said bottom surfaceof said membrane, said first and second microfluidic channels eachcomprising a surface that is parallel to said membrane, and eachcomprising side walls. It is not intended that the method be limited toseeding only intestinal cells. In one embodiment, hypothalamic neuronsare in said second microfluidic channel. While not limited to anyparticular cell position, in one embodiment, said hypothalamic neuronsgrow on the parallel surface and side walls of the second microfluidicchannel so as to form a lumen. A variety of biomarkers can be assessed.In one embodiment, said intestinal cells express the marker E-Cadherin.It is not intended that the present invention be limited to anyparticular amount of storage; storage can be for days, weeks, months ormore. In one embodiment, said storing of said selected cells in step c)is performed for at least one month. Similarly, it is not intended thatthe present invention be limited to any precise period of time forculturing. In one embodiment, said culturing of step f) is performed forat least four days, more typically seven days, or ten days, or fourteendays or more. The microfluidic device can have additional features. Forexample, in one embodiment, said microfluidic device further comprisesat least one inlet port and at least one outlet port, and said culturemedia enters said inlet port and exits said outlet port.

As indicated above, this embodiment of the method contemplates b)subjecting said organoid cells to a selection reagent to generateselected cells. In one embodiment, said selected cells comprise foregutprogenitors, midgut progenitors and/or hindgut progenitors. In oneembodiment, said organoids are derived from human induced pluripotentstem cells. In one embodiment, said organoids are derived from inducedpluripotent stem cells from a human patient diagnosed with agastrointestinal disorder. In one embodiment, said induced pluripotentstem cells are from a patient diagnosed with Inflammatory bowel disease(IBD). In one embodiment, said induced pluripotent stem cells are from apatient diagnosed with colitis.

In yet another embodiment, the present invention contemplates a method,comprising: a) differentiating induced pluripotent stem cells (iPSCs)into gastrointestinal organoids (iGIOs) and hypothalamic neurons (iHTNs)cells; and b) seeding said cells on an organ-on-chip microfluidicdevice. In one embodiment, said organoids comprise foregut progenitorcells, midgut progenitors and/or hindgut progenitor cells. In oneembodiment, the method further comprises c) culturing said seeded cellsunder flow conditions that support the maturation and/or differentiationof said seeded cells from said organoids into intestinal cells. In oneembodiment, said organoids are derived from induced pluripotent stemcells from a human patient diagnosed with a gastrointestinal disorder.In one embodiment, said induced pluripotent stem cells are from apatient diagnosed with Inflammatory bowel disease (IBD). In oneembodiment, said induced pluripotent stem cells are from a patientdiagnosed with colitis. In one embodiment, said organoids are derivedfrom induced pluripotent stem cells from a human with an abnormal bodymass index. In one embodiment, said body mass index is greater than 50.In one embodiment, cells were selected from said organoids and werestored frozen and then thawed prior to step b). Again, a variety ofmicrofluidic device designs are contemplated. In one embodiment, saidorgan-on-chip microfluidic device comprises a membrane, said membranecomprising a top surface and a bottom surface, and wherein cells fromsaid organoids are seeded on said top surface and said neurons areseeded on said bottom surface. In one embodiment, said organ-on-chipmicrofluidic device further comprises a first microfluidic channel influidic communication with said top surface of said membrane and asecond microfluidic channel in fluidic communication with said bottomsurface of said membrane, said first and second microfluidic channelseach comprising a surface that is parallel to said membrane, and eachcomprising side walls. In one embodiment, said neurons are present onthe parallel surface and side walls of the second fluidic channel so asto constitute a lumen.

In yet another embodiment, the present invention contemplates a method,comprising: a) providing i) a microfluidic device, ii) intestinal cellsand iii) hypothalamic neurons; and b) seeding said cells on saidmicrofluidic device. In one embodiment, said intestinal cells areprimary cells. In another embodiment, said intestinal cells are derivedfrom stem cells (e.g. said stem cells are induced pluripotent stem cells(iPSCs). In one embodiment, the method further comprises c) culturingsaid seeded cells under flow conditions that support the maturationand/or differentiation of said seeded cells.

In addition to methods, the present invention contemplates kits andsystems. Kits can provide a microfluidic device and the organoid cells(fresh or frozen), along with instructions on how to seed the cells ontothe device. The systems can involve a number of components. For example,in one embodiment, the system comprises a) a fluidic device comprising amembrane, said membrane comprising a top surface and a bottom surface,said top surface comprising primary intestinal cells or stemcell-derived intestinal cells, said microfluidic device furthercomprising a first fluidic channel in fluidic communication with saidtop surface of said membrane and a second fluidic channel in fluidiccommunication with said bottom surface of said membrane, b) a fluidsource in fluidic communication with said first and second fluidicchannels, whereby said cells are exposed to fluid at a flow rate. Thesystem is not limited to just cells of one type. In one embodiment, thesystem further comprises iPSC-derived neurons (and in particular,iPSC-derived neurons that are hypothalamic neurons). In one embodiment,the stem cell-derived intestinal cells and the iPSC-derived hypothalamicneurons are generated from the stem cells of the same person. In anotherembodiment, the stem cell-derived intestinal cells and the iPSC-derivedhypothalamic neurons are generated from the stem cells of differentpeople. In one embodiment, the stem cell-derived intestinal cells arefrom a human patient diagnosed with a gastrointestinal disorder. In oneembodiment, the stem cell-derived intestinal cells are from a patientdiagnosed with Inflammatory bowel disease (IBD). In one embodiment, thestem cell-derived intestinal cells are from a patient diagnosed withcolitis. In one embodiment, the stem cell-derived intestinal cells arederived from a human with an abnormal body mass index. In oneembodiment, said body mass index is greater than 50.

The present invention also contemplates methods of populating amicrofluidic device with intestinal cells, comprising disaggregatinghuman intestinal organoids (HIOs) into single cells; and adding thesingle cells to the device. The device can have a number of designs(e.g. one or more channels, one or more membranes, etc.). In oneembodiment, the single cells are purified based on CD326+ expressionbefore addition to the apparatus. In one embodiment, adding the singlecells to the apparatus comprises resuspension in a media comprising oneor more of: ROCK inhibitor, SB202190 and A83-01. In one embodiment, theHIOs are cultured in the presence of ROCK inhibitor prior todisaggregation. In one embodiment, the HIOs are derived from inducedpluripotent stem cells (iPSCs). In one embodiment, the iPSCs arereprogrammed lymphoblastoid B-cell derived induced pluripotent stemcells (LCL-iPSCs). In one embodiment, the iPSCs are reprogrammed cellsobtained from a subject afflicted with an inflammatory bowel diseaseand/or condition. In one embodiment, derivation of HIOs from iPSCscomprises: generation of definitive endoderm by culturing iPSCs in thepresence of Activin A and Wnt3A; differentiation into hindgut byculturing definitive endoderm in the presence of FGF and either Wnt3A orCHIR99021; collection of epithelial spheres or epithelial tubes;suspension of epithelial spheres or epithelial tubes in a gel matrix(e.g. Matrigel); and culturing in the presence of one or more growthfactors (e.g. CHIR99021, noggin and EGF). In a preferred embodiment, theintestinal cells form an organized structure comprising villi. In oneembodiment, the villi are lined by one or more epithelial cell lineagesselected from the group consisting of: absorptive, goblet,enteroendocrine, and Paneth cells. In one embodiment, the organizedstructure possesses barrier function, cytochrome P450 activity, and/orapical mucus secretion.

The present invention also contemplates devices, such as microfluidicdevices comprising: a population of intestinal cells, wherein thepopulation comprises an organized structure. In a preferred embodiment,the organized structure comprises villi. In one embodiment, the villiare associated with or lined by one or more epithelial cell lineagesselected from the group consisting of: absorptive, goblet,enteroendocrine, and Paneth cells. In one embodiment, the organizedstructure possesses barrier function, cytochrome P450 activity, and/orapical mucus secretion. In one embodiment, the intestinal cells arederived from human intestinal organoids (HIOs) disaggregated into singlecells and purified based on CD326+ expression. In one embodiment, theHIOs are derived from iPSCs by a method comprising: generating adefinitive endoderm by culturing iPSCs in the presence of Activin A andWnt3A; differentiating the endoderm into hindgut by culturing definitiveendoderm in the presence of FGF and either Wnt3A or CHIR99021;collecting epithelial spheres or epithelial tubes; suspending theepithelial spheres or epithelial tubes in a gel matrix (e.g. Matrigel);andculturing in the presence of one or more growth factors (e.g.CHIR99021, noggin and EGF).

DEFINITIONS

For purposes of the present invention, the following terms are definedbelow.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise. Also, as used in the descriptionherein, the meaning of “in” includes “in” and “on” unless the contextclearly dictates otherwise.

As used herein “gastrointestinal” (GI) or “gastrointestinal tract” or“gut” in reference to an “intestinal” cell refers to any cell found inany region of the GI tract and differentiated cells with biochemicaland/or structural properties akin to cells found in the GI tract.Regions of the GI include the foregut, midgut and hindgut regions. Thus,intestinal cells can be from each of these regions with differentiatedcells possessing foregut-like, midgut-like, and hindgut-like properties.The present invention contemplates “intestinal cells” to be cells thatare part of the GI tract structure, e.g. stomach cells, small intestinecells, intestinal epithelial cells, secretory cells, endocrine cells,nerve cells, muscle cells, stromal cells, etc.

The term lumen refers to a structure having an inner open space, such asa central cavity of a tubular or hollow structure. As one example, aninner open space surrounded by cells forming a tube. The tube need notbe circular. Thus, when cells grow on all sides of a microfluidicchannel there can be a lumen.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1A-F: Human iPSCs Differentiate into Endocrinally Active ForegutEpithelium (iFGE) by Modulation of WNT, FGF, BMP and Retinoic AcidSignaling. (FIG. 1A) A schematic of an exemplary Foregut epithelium(iFGE) differentiation protocol. (FIG. 1B) RT-qPCR of foregut genesshown to be significantly increased (** p<0.01) in the Inventors' Day 20iFGE compared to Day 0, ND: Not detectable. Two-way ANOVA was employedto determine differences within Day 0 and Day 20 iFGEs (FIG. 1C) Brightfield images of Day 6 and Day 20 iFGE. (FIG. 1D) Panel showing foregutepithelial markers E-cadherin (CDH1), β-catenin (CTNNB) and endoderm andforegut progenitors Sox2 and Sox17; (FIG. 1E) Panel showing expressionof neuroendocrine markers such as synaptophysin (SYP), Somatostatin andSerotonin; (FIG. 1F) Panel (top to bottom) showing gastric endocrinepositive cells such as ghrelin, peptide YY and gastrin. Data shown hereare representative of average results from the two iPSC linesdifferentiated multiple times in independent experiments.

FIG. 2A-O: Functional Neuropeptidergic Hypothalamic Neurons (iHTNs) canbe Derived from hiPSC-Neuroepithelium by Activating SHH and InhibitingWNT Signaling. (FIG. 2A) A schematic of an exemplary Hypothalamic neuron(iHTN) differentiation protocol. (FIG. 2B) RT-qPCR of hypothalamic andarcuate nucleus specific genes showing significantly increasedexpression of the genes at day 40 of differentiation compared to Day 0(*p<0.05, ** p<0.01). ND: not detectable; Two-way ANOVA was employed todetermine differences within Day 0 and Day 40 iHTNs (FIG. 2C)Measurement of hypothalamus-specific neuropeptide Y (NPY) measured fromcell supernatants using ELISA (p<0.001 determined using paired t-test).(FIG. 2D) Measurement of hypothalamus-specific α-melanocyte stimulatinghormone (α-MSH) measured from cell supernatants using ELISA (*** p<0.001determined using paired t-test). (FIG. 2E-FIG. 2N) panel showsimmunopositivity for hypothalamic progenitors and neuropeptidergicmarkers. (FIG. 2O) MEA readings of neurons from Day 0 as well as Day 40from the same electrode over time showing increased neuronal firing inDay 40 neurons. Images and data shown here are representative of averageresults from the two iPSC lines differentiated multiple times inindependent experiments.

FIG. 3A-F: Chronic Low-Dose EDC Treatment Perturbs NF-κB signaling iniFGEs and iHTNs Without Affecting Cell Viability. (FIG. 3A) A schematicrepresentation of EDC treatments and analysis performed on iFGEs andiHTNs. (FIG. 3B) EDC treatment schematic showing the treatment planscarried out on iFGEs and iHTNs. (FIG. 3C). Immunoctochemistry showingincrease in phospho p65 (red) (*** p<0.001) in iFGE co-stained withghrelin (green). (FIG. 3D) immunocytochemistry revealing increasedphospho p65 (red) (*** p<0.001) in iHTN co-stained with Synaptophysin(green). (FIG. 3E) Representative Western blots and quantified bargraphs show an increase in phospho p65 protein levels in iFGE, ***p<0.001. (FIG. 3F) Representative western blots and quantified bargraphs show an increase in phospho p65 protein levels in iHTN (n=4), **p<0.01. MTT assay showing no significant differences in cell viabilityin any EDC treatment in both iFGE and iHTN respectively. All statisticalanalysis performed using one-way ANOVA. Data shown are representative ofaverage results from the two iPSC lines differentiated n =3 times inindependent experiments.

FIG. 4A-F: EDC treatment shows increases in Canonical and Non-canonicalPathway.

(FIG. 4A & FIG. 4D) FIG. 4A) schematic representation of NF-κB canonicaland non-Canonical pathways. (FIG. 4B & FIG. 4E) Representative Westernblots and quantified bar graphs showing increases in p50 and p52 levelsin iFGE, *** p<0.001, (FIG. 4C & FIG. 4F) Representative Western blotsand quantified bar graphs showing increases in p50 and p52 levels iniHTN (n=4), *** p<0.001. All statistical analysis performed usingone-way ANOVA.

FIG. 5A-F: EDCs Impinge on Metabolic Activity by DisruptingMitochondrial Respiration. (FIG. 5A-FIG. 5B) Seahorse assay measurementsof mitochondrial respiration with quantified bar graphs representingchanges in spare respiratory capacity in iFGE and iHTN respectively, *p<0.05; **p<0.01; EDCs decrease expression of both nuclear andmitochondrially-encoded respiratory genes in iFGEs. RT-qPCR relativenormalized expression of nuclear (SCO2, POLRMT, TFAM) andmitochondrial-encoded (CYTB5) genes involved in mitochondrialrespiration from iFGEs (FIG. 5C-FIG. 5D). (FIG. 5C) RT-qPCR showing mRNAlevels of mitochondrial genes encoded by nucleus SCO2, POLRMT. (FIG. 5D)mRNA levels of nuclear encoded mitochondrial gene TFAM andmitochondrially encoded gene CYB5A, also decreased upon EDC treatment ofiFGEs. *p<0.05, **p<0.01, ***p<0.001. n=3. and iHTNs (FIG. 5E-FIG. 5F).EDC treatment significantly decreased expression of these genes *p<0.05, ** p<0.01, *** p<0.001. ND: Not detectable. All statisticalanalysis performed using one-way ANOVA.

FIG. 6A-D: NF-κB Inhibition Rescues Cells from NF-κB Pathway Activationand Mitochondrial Impairment in Human Foregut Epithelium. (FIG. 6A)Immunoblots show exemplary NF-κBi treatment decreases EDC mediatedincreases in Phospho p65, p50, and p52, *** p<0.001. 2 different celllines were loaded in 6 lanes as Lane 1,2 and 3 belonging to 80iCTR (Vh1,Comb1 and NFκBi1) and lanes 4,5 and 6 from 201iCTR (Vh2, Comb2 andNFκBi2). (FIG. 6B) Immunocytochemistry showing phosphor p65 staining invehicle treatment (Vh), increased phosphor p65 with EDC combinationtreatment (Comb) which decreases with NF-κBi, *** p<0.001. (FIG. 6C)Seahorse assay showing improved mitochondrial respiration upon NF-κBitreatment compared to combination treatment, ** p<0.01. (FIG. 6D)RT-qPCR expression levels of SCO2, POLRMT, TFAM and CYTB5 showingdecreased mitochondrial respiratory genes with combination treatmentwhich are rescued by NF-κBi treatment, * p<0.05, ** p<0.01, ***p<0.001.All statistical analysis performed using one-way ANOVA.

FIG. 7A-D: NF-κB Inhibition Rescues Cells from NF-κB Pathway Activationand Mitochondrial Impairment in Human Hypothalamic Neuron Cultures.(FIG. 7A) Immunoblots show exemplary NF-κBi treatment decreases EDCmediated increases in Phospho p65, p50, and p52, * p<0.05. 2 differentcell lines were loaded in 6 lanes as Lane 1,2 and 3 belonging to 80iCTR(Vh1, Comb1 and NFκBi1) and lanes 4,5 and 6 from 201iCTR (Vh2, Comb2 andNFκBi2). (FIG. 7B) Immunocytochemistry showing phospho p65 staining invehicle treatment (Vh), increased phosphor p65 with EDC combinationtreatment (Comb) which decreases with NF-κBi, * * * p<0.01. (FIG. 7C)Seahorse assay showing improved mitochondrial respiration upon NF-κBitreatment compared to combination treatment, ** p<0.001. (FIG. 7D)RT-qPCR expression levels of SCO2, POLRMT, TFAM and CYTB5 showingdecreased mitochondrial respiratory genes with combination treatmentwhich are rescued by NF-κBi treatment, * p<0.05, ** p<0.01, ***p<0.001.All statistical analysis performed using one-way ANOVA.

FIG. 8A-F: Characterization of PBMC-derived iPSCs. (FIG. 8A) Schematicrepresentation depicting the episomal reprogramming and generation ofiPSCs. (FIG. 8B) Bright-field images of the reprogrammed iPSC coloniesfrom 2 control lines (80iCTR and 201iCTR) which show high alkalinephosphatase activity and immunopositivity for pluripotency surfacemarkers such as SSEA, OCT4, TRA-1-60, NANOG, TRA-1-81 and SOX2. (FIG.8C) Gene chip- and bioinformatics PluriTest characterization of the 2control lines. (FIG. 8D) G-band karyotyping showing normal phenotypes ofboth cell lines. (E) qPCR of both iPSC lines showing clearance of thereprogramming plasmids. (FIG. 8F) Agarose gel electrophoresis showingthe absence of EBNA factor in the two iPSC lines.

FIG. 9A-G: MTT assay determining EDC dose response. Exemplary graphsshowing dose response to half log doses of (FIG. 9A) PFOA, (FIG. 9C) TBTand (FIG. 9E) BHT. The highlighted dose has been used in this study. Bargraphs representing the optical density values of MTT assay on iHTNstreated with increasing doses of (FIG. 9B) PFOA, (FIG. 9D) TBT (FIG. 9F)BHT and (FIG. 9G) Mt DNA assay as a long-range PCR DNA damage assayshowing lack of mitochondrial DNA lesions with EDC treatment. Note: Aslight increase in nuclear HPRT and Average nuclear lesions was observedwith TBT and combination treatment alone. *p<0.05; ***p<0.001. n=3.

FIG. 10A-E: iFGE differentiation efficiency and full immunoblots.Original images of iFGE immunoblots represented in FIG. 3A-F and 4A-F.(FIG. 10A) ICC quantification of E-cadherin positive cells in our iFGEcultures showing no differences in epithelium forming capacity betweenuntreated and EDC-treated conditions; (FIG. 10B, FIG. 10C, FIG. 10D,FIG. 10E). Full immunoblots of iFGE samples represented in FIG. 4A-F.

FIG. 11A-F: Intact p53 protein expression in differentiated iHTNs, EDCtreatment does not effect iHTN differentiation efficiency and full iHTNimmunoblots. Original images of iHTN immunoblots represented in FIG.3A-F and 4A-F. (FIG. 11A) Day 40 iHTNs showing expression of total p53protein in 201iCTR and 80iCTR. (FIG. 11B) Quantification of OTP+/TuJ1+cells in iHTN differentiation. (FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F)Original images of iHTN immunoblots represented in FIG. 4A-F.

FIG. 12A-B: Cox IV densitometry as measures of equal mitochondrial mass.Exemplary graphs showing Cox IV densitometry revealing equal amounts ofcytochrome C oxidase 4 used as loading controls and as measures ofmitochondrial mass in the samples employed. Cox IV densitometryrevealing equal amounts of cytochrome C oxidase 4 in (FIG. 12A) iFGEsand (FIG. 12B) iHTNs used as loading controls and as measures ofmitochondrial mass in the samples employed.

FIG. 13A-C: Cox IV densitometry as measures of equal mitochondrial mass.Original images of iFGE blots and threshold-based quantification. (FIG.13A) Western blots in iFGEs showing no rescue of ER stress markers uponNFκBi treatment compared to EDC-treated conditions (FIG. 13B) Originalimages of iFGE blots represented in FIG. 18A-H. 2 different cell lineswere loaded in 6 lanes as Lane 1,2 and 3 belonging to 80iCTR (Vh1, Comb1and NFκBi1) and lanes 4,5 and 6 from 201iCTR (Vh2, Comb2 and NFκBi2).(FIG. 13C) Quantification of immunocytochemistry staining of phosphoNF-κB p65 in iFGEs using MetaXpress with the threshold tool to measurespecific Phospho p65 signals. The panel represents images postthresholding in each of the treatments. n=3.

FIG. 14A-C: Original images of iHTN blots and threshold-basedquantification. (FIG. 14A) Immunoblots showing exemplary no rescue inphospho p53 (Ser15) levels upon NF-κBi treatment compared to EDC-treatedconditions. *p<0.05. (FIG. 14B) Original images of iHTN blotsrepresented in FIG. 19. 2 different cell lines were loaded in 6 lanes asLane 1,2 and 3 belonging to 80iCTR (Vh1, Comb1 and NFκBi1) and lanes 4,5and 6 from 201iCTR (Vh2, Comb2 and NFκBi2). (FIG. 14C) Quantification ofimmunocytochemistry staining of phospho NF-κB p65 in iHTNS usingMetaXpress with the threshold tool to measure specific Phospho p65signals. The panel represents images post thresholding in each of thetreatments. n=3.

FIG. 15A-B: Chronic Low-Dose EDC Treatment ER stress in iFGEs and iHTNsWithout Affecting Cell Viability. (FIG. 15A and FIG. 15B) Representativeimmunoblots showing levels of bona fide ER stress pathway proteins,IRE1, BiP and Ero1, in (FIG. 15A) iFGE and (FIG. 15B) iHTNs. Quantifiedhistograms using ImageJ-based densitometry of bands for each of therespective protein immunoblots normalized to Cox IV as loading controlare shown below and represented as fold-change compared tovehicle-treated control. IRE1 protein increases, while BiP and Ero1levels decrease in response to EDC exposure, *p<0.05, ** p<0.01, ***p<0.001. All statistical analysis was performed using one-way ANOVA.Data shown are representative of average results from the two iPSC linesdifferentiated n=3 times in independent experiments. This informationsupplements FIG. 3A-F.

FIG. 16A-C: EDC treatment causes disturbances in NF-κB p65 Canonical andNon-canonical Pathways. (FIG. 16A) Top panel: Representativeimmunocytochemistry (ICC) showing increases in phosphorylated p65 (red)in iFGEs co-stained with ghrelin (green); Bottom panel: RepresentativeICC showing increases in phosphorylated p65 (red) in iHTNs co-stainedwith synaptophysin (green). (*** p<0.001). Immunopositive cells werescored and quantified inhistograms for both iFGEs and iHTNs, which isrepresented by fold-change in phosphorylated NF-κB p65immunopositivecells in each of the EDC treatments compared to the vehiclecontrol-treated iFGEs (*** p<0.001) and iHTNs (*** p<0.001).Representative immunoblots for protein levels in whole cell lysateshowing increases in phosphorylated p65, total p50 and total p52 levelsin (FIG. 16 B) iFGE, *** p<0.001 and FIG. 16C) iHTNs *** p<0.001.Quantified histograms using ImageJ-based densitometry of bands for eachof the respective immunoblots are shown below and represented asfold-change compared to vehicle-treated control. Ratio of phosphorylatedNF-κB p65 over total p65, p50/105 (canonical) and p52/p100(non-canonical) were calculated. All statistical analysis were performedusing one-way ANOVA. Images and data shown are representative of averageresults from the two iPSC lines differentiated n=3 times in independentexperiments. This information supplements FIG. 4A-F.

FIG. 17A-G: EDCs Induce Metabolic Stress and Disrupt EndocrineRegulation. (FIG. 17A) Immunoblots showing exemplary decreases inphosphorylated p53 (Ser15) in both iFGE and iHTN (*** p<0.001) upon EDCexposure, (FIG. 17B) Seahorse mitochondrial respirometry measurements ofwith histograms representing changes in spare respiratory capacity iniFGE and iHTN, * p<0.05; **p<0.01; (FIG. 17C) RT-qPCR relativenormalized expression of nuclear (SCO2, POLRMT, TFAM) andmitochondrial—encoded (CYB5A) genes involved in mitochondrialrespiration from iHTNs. (FIG. 17D) Putative binding motifs for NF-κB p65(RelA) and p53 transcription factors on the DNA of SCO2, POLRMT, TFAM,CYB5A, TP53, and RELA genes shown in the table displays number ofpossible binding sites and distance from transcription start site at aconfidence level of 70%; Red fonts IL1A and CDKN1A are known to bepositively regulated genes by p65 and p53 respectively, (FIG. 17E)Measurement of ATP levels (ATP/ADP ratio) showing decreases withEDC-treatments, (FIG. 17F) Immunoblots showing decreases in PYYlevels inEDCs treated iFGEs; (FIG. 17G) ELISA of α-MSH showing decreases insecretion with EDC treatment of iHTNs. * p<0.05, ** p<0.01, *** p<0.001,n=3. ND: Not detectable. All statistical analysis was performed usingone-way ANOVA. Data shown are representative of average results from thetwo iPSC lines differentiated n=3 times in independent experiments. Thisinformation supplements FIG. 5A-F.

FIG. 18A-H: Blocking NF-kB Rescues EDC-mediated Metabolic Stress &Endocrine Dysfunction. Immunoblots showing exemplary NF-kBi treatmentdecreases EDC-mediated increases in phosphorylated p65, p50, and p52 in(FIG. 18A) iFGEs and (FIG. 18B) iHTNs, *p<0.05, **p<0.01, *** p<0.001.Two different cell lines were loaded in 6 lanes with lanes 1, 2 and 3belonging to80iCTR (Vh1, Comb1 and NF-κBi1) and lanes 4, 5 and 6 from201iCTR (Vh2, Comb2 and NF-κBi2). (FIG. 18C) Immunocytochemistry showingphosphorylated p65 staining in vehicle treatment (Vh), increasedphospho-p65 with EDC combination treatment (Comb) that decreases withNF-κBi, * p<0.05, **p<0.01, *** p<0.001. (FIG. 18D) Seahorse assayshowing improved mitochondrial respiration upon NF-κBi treatmentcompared to combination treatment in iHTNs, *** p<0.001. (FIG. 18E)RT-qPCR expression levels of SCO2, POLRMT, TFAM and CYB5A showingdecreased mitochondrial respiratory genes with combination treatmentthat are rescued by NF-κBi treatment, * p<0.05, ** p<0.01, ***p<0.001.(FIG. 18F) Restoration of ATP levels upon NF-κBi treatment, **p<0.01,***p<0.001; (FIG. 18G) α-MSH secretion levels showed improvement uponNF-κBi treatment, ***p<0.001, (FIG. 18H) Western blot showing rescue ofPYY levels in iFGEs, * p<0.05, **p<0.01. All statistical analysis wasperformed using one-way ANOVA. Images and data shown are representativeof average results from the two iPSC lines differentiated n=3 times inindependent experiments. This information supplements FIGS. 5A-F and6A-D.

FIG. 19: Proposed model of EDC-mediated dysregulation in developingendocrine cells. A schematic diagram of a cell showing a proposed modelof EDC-mediated dysregulation in developing pluripotent stemcell-derived endocrine tissues. Developing endocrine cells when exposedto EDCs such as PFOA, TBT and BHT trigger endoplasmic reticulum (ER)stress by increasing IRE1 and downregulation of Ero1 and BiP, which areknown to induce an unfolded protein response (UPR) in a cell. Thisresults in perturbation of NF-κB (increased phosphorylation of p65) andp53 (decreased phosphorylation of p53 at Ser15) signaling in parallel.The subsequent metabolic stress is comprised of reduced transcription ofboth nuclear- and mitochondrial-encoded respiratory genes, defectivemaximal respiration and mitochondrial spare respiratory, and a decreasein cellular bioenergetics/ATP levels. Intricate crosstalk betweenunhealthy mitochondria and ER may further lead to ER stress in afeedback loop and thereby exacerbate this mechanism. Overall, bothaccumulations of misfolded proteins as well as a decrease in ATP levelsupon chronic exposure to low-dose of EDCs induces metabolic stress in anendocrine cell, thereby negatively impacting endocrine regulation due toabnormal production and secretion of gut and brain neuropeptides.

FIG. 20A-D: Bioinformatic determination of putative DNA binding sitesfor NFκB-p65 (RELA) and TP53. (FIG. 20A) Charts showing identificationof the number of putative binding sites of NFκB-p65 and TP53 bindingmotifs on genes of interest such as SCO2, POLRMT, TFAM, CYB5A andrespective known genes regulated by NFKB-p65 (RELA) such as IL1A, IL1B,TNF, IL6 or regulated by TP53 such as GADD45A, GADD45B, GADD45G, PERP,BAX. (FIG. 20B) Identification of minimum distance in base pairsupstream of the transcription start sites of the DNA binding motifs ofNFκB-p65 and TP53 on the indicated genes of interest. HOX genes wereemployed as neutral genes or genes that are not well-known in theliterature to be controlled either by NFκB-p65 and TP53. DNA bindingmotif as a sequence logo graphical representation of the sequenceconservation of nucleotides where the sixe of the nucleotide letterrepresents the frquecny of the letter at that position in the sequencefor (FIG. 20C) NFκB-p65 and (FIG. 20D) TP53 used in the bioinformaticanalyses.

FIG. 21: Stomach (foregut) optimization on chips. A schematic timelineshowing exemplary 3D organoid maturation from endoderm for an exemplaryForegut—stomach differentiation protocol. iFG-MO=Day 6 mini organoids;Epi-iFG=Day 6 mini organoids sorted on Day 20. Epi-iFG=Day 6 miniorganoids sorted on Day 20. iFG-O-diss=Day 34 organoids dissociated.

FIG. 22A-I: Characterization of D34 iFG-O (organoid) by ICC. Fluorescentmicrographs of cells and tissues stained with exemplary immunomarkersfor immunocytocheistry (ICC) characterization of the cells/tissues usedfor seeding chips. Examples of markers, FIG. 22A) E-cadherin (red); FIG.22B) Sox2 (green); FIG. 22C) Muc5AC (red); FIG. 22D) Synaptophysin(red); FIG. 22E) Serotonin; FIG. 22F) Somatostatin (green); FIG. 22G)Gastrin (green); FIG. 22H) Ghrelin (red); and FIG. 22I) Peptide YY(red). Inserts in FIG. 22G and FIG. 22H are enlarged areas outlined bythe smaller boxes.

FIG. 23: Overall Plan for cells to be used for seeding foregut on achip. A schematic timeline showing endoderm induction and foregutdifferentiation of iPSCs within increasing amounts of fetal bovine serum(FBS) in the presence of Activin A and Wnt3A followed by the addition ofCHIR, FGF4, LDN, and RA at day 3 onwards. iFG-O-diss=Day 34 organoidsdissociated; iFG-MO=Day 6 mini organoids; Epi-iFG=Day 6 mini organoidssorted on Day 20. Epi-iFG=Day 6 mini organoids sorted on Day 20.iFG-O-diss=Day 34 organoids dissociated.

FIG. 24: Stomach-hypothalamus co-culture on a chip. An exemplaryschematic of one embodiment of a microchip. This chip shows iFG-MO cellsin the upper channel with iHTN in the lower channel. Goal: To test ifthe presence of hypothalamic neurons (iHTNs) can be co-cultured on achip. Approach: Apical channel was seeded with iFG-MO and the basalchannel with iHTNs. Co-culturing foregut with iFG-MO (mo: minoorganoids)with induced hypothalamic neurons (iHTNs). We also decreased flow rateto 10 uL/hr due to over proliferation of iFG-MO in the previous set ofexperiments.

FIG. 25A-D: Confocal images of fluorescing markers. Exemplaryimmunofluorescent micrographs of cells on chips stained withimmunofluorecent markers in upper and lower channels of chips. FIG. 25A)All fluorescent channels showing immunofluorescence emitting from upperand lower channels of the chip. FIG. 25B) Sox2 fluorescence observed onapical region. FIG. 25C) E-cadherin fluorescence observed on apicalregion. FIG. 25D) Tun fluorescence observed on basal region. Imagesshowing markers in respective channels and regions (see previous Figurefor exemplary cells in upper and lower channels) under flow (10 ul/hr).The markers were very specific and were found only in their respectivechannels.

FIG. 26A-C: Confocal imaging of IFG-MO on Day 21 under flow (30 ul/hr).Exemplary immunofluorescent micrographs of cells in chips stained withimmunofluorecent markers. FIG. 26A) Foregut progenitor cells stainedwith DPAI and SOX2. FIG. 26B) Endocrine cells stained with SYP. And FIG.26C) Epithelium stained with E-cadherin.

FIG. 27A-B: iFG-MO seeded on apical channel. Flow (10 ul/hr). Exemplaryimmunofluorescent micrographs of cells in chips stained withimmunofluorecent markers. FIG. 27A) Fewer Sox2+ and FIG. 27B) Highernumbers of SYP+ cells in comparison to cells grown under 30 ul/hr flowrates.

FIG. 28: Optimizing foregut epithelium. An exemplary schematic of oneembodiment of a microchip along with a schematic timeline for foregutand organoid maturation. Goal: To optimize the formation of foregutepithelium by a better and more streamlined selection of Day 6 organoidsusing a Selection reagent, described herein. Approach: Apical channelseeded with iFG-SR by selecting organoids using a selection reagent.Maintain decreased flow rate at 10 uL/hr. Decrease EGF concentration inmedium gradually to encourage differentiation and maturation. At thispoint the selection of Day 6 organoids came out to be a crucial step inobtaining good epithelium, based on some experiments performed in thelab and hence we tried a selection reagent which effectively separatecell clusters from the surrounding monolayer and appeared to be aneffective way to pick Day 6 organoids for plating.

FIG. 29: Exemplary Experimental Timecourse showing lowering amounts ofan agent. A schematic timeline showing iFG-SR cells grown underdecreasing amounts of a maturation agent, e.g. EGF.

FIG. 30: Exemplary general characterization of the tissue used forseeding chips. Exemplary immunofluorescent micrographs of cells on chipsstained with immunofluorecent markers, e.g. E-cadherin, Sox2, Sox17,synaptophysin, serotonin, somatostatin, gastrin, ghrelin, and peptideYY. Characterization of D20 iFG-SR cells by ICC on a 96-well plate (2DDay 20).

FIG. 31A-B: Comparative tile scan images of iFG-SR and iFG-MO stainedfor E-cadherin. Exemplary immunofluorescent micrographs of cells onchips stained with an immunofluorecent marker for E-cadherin. FIG. 31A)iFG-SR and FIG. 31B) iFG-MO. Under flow rate of 10 ul/hr.

FIG. 32: Ghrelin secretion by ELISA comparing SR and hand picked D6O.Exemplary bar graph of ghrelin secretion from cells grown on chips.Several exemplary cultures of iFG-SR and iFG-MO were compared forghrelin secretion (pg/mg of cell protein) from day 15-22 and day 23-30of chip culture.

FIG. 33: Comparison of our foregut system with a positive control(NCI-N87 gastric cancer line). An exemplary schematic of one embodimentof a microchip along with a schematic timeline for foregut and organoidmaturation including a selection reagent and decreasing amounts of EGF.Goal: To compare iFG-SR to human gastric cancer (HGC)(NCI-N87-epithelial) line. Approach: Apical channel seeded with iFG-SRor HGC. Maintain decreased flow rate at 10 uL/hr. Compare the 2 celltypes on chips by ICC and Ghrelin secretion. The HGC line is maintainedin their optimal growth medium with no variations throughout theexperiment. At this point the selection of Day 6 organoids came out tobe a crucial step in obtaining good epithelium, based on someexperiments performed in the lab and hence we tried a selection reagentwhich effectively separate cell clusters from the surrounding monolayerand appeared to be an effective way to pick Day 6 organoids for plating.

FIG. 34A-B: Flow Conditions On HGC and iFG-SR Chips. Micrographs of celllayers in chips under flow conditions comparing inmmunofluorescentstaining of SOX2, SYP and E-cadherin (E-cad) between FIG. 34A) HGC andFIG. 34B) iFG-SR cells.

FIG. 35: Comparative Tile Scan of HGC and iFG-SR cell layers. Exemplarycomparative micrographs of cell layers comparing iRG-SR and HGC growingwith and without flow conditions in chips. Flow worked better for iFG-SRbut not for HGC. iFG-SR epithelium looked better under no flowconditions than under flow movement.

FIG. 36: Steady increase in Ghrelin secretion with flow in iFG-SR. Anexemplary bar graph showing iFG-SR cell production of ghrelin secretionof cells in chips under flow chip conditions compared to lower amountsfrom cells in no flow chips.

FIG. 37: Exemplary experimental flowchart and set up. A schematictimeline showing an exemplary chip, experimental conditions and examplesof assays. iPSC derived Stomach organoids and iFG-MO seeded to theapical channel; iHTNs seeded on the basal channel for functional assayand imaging; growth of iHTNs in chip and imaging for foregut andneuronal markers such as Sox2, E-cadherin and TuJ1. Cultured induplicate under no flow and flow conditions (Flow 10 uL/hr).

FIG. 38: One embodiment of an “Organ on chip” microfluidic device. Anexemplary diagram illustrating the difference between static transwellculture, FIG. 38B, of gastrointestinal organoids (iGIOs) andhypothalamic neurons (iHTNs), which were differentiated from iPSCs, andculture under flow conditions in “organ on chip” microfluidic devices,FIG. 38A.

FIG. 39A-G: Exemplary Results Using An “Organ on chip” MicrofluidicDevice Of The Previous Figure. Provides exemplary experimental resultsof immunostaining of cells using an organs-on-a-chip model of iGIOs andiHTNs. FIG. 39A) Shows a chip with apical (Red) and basal (Blue)channels. FIG. 39B) shows iGIOs differentiated on the apical channel.FIG. 39C) Shows GI epithelium on chip that is E-cadherin+(white) withSox2+ foregut progenitors (green). FIG. 39D) Shows iGIOs on chip showingepithelium (white) and synaptophysin+endocrine cells (red). FIG. 39E) isa confocal 3D image of seeded chip with iHTNs in basal channel (Tuj-1,staines Neuron-specific class III β-tubulin), while FIG. 39F and FIG.39G show SOX2+ (SRY-Box 2) foregut, and E-cadherin+ epithelium in apicalchannel (respectively). White arrows point to the porous membranewhile * identifies a lumen surrounded by neuronal cells in FIG. 39E-FIG.39F.

FIG. 40: Gut-On-Chip. Shows an illustrative schematic of one embodimentof a small microfluidic device illustrating upper and lower chambersseparated by a porous membrane. Arrows represent continuous flow ofmedia in both upper and lower channels. Gut epithelium is on top of theporous membrane in an upper channel. Vacumm chambers are located on theoutside of both sides of the channel areas.

FIG. 41: Shows an exemplary micrograph of organoids. Intestinalorganoids were grown and used for embodiments of microfluidic chipsdescribed herein.

FIG. 42A-D: Shows fluorescently stained micrographs of intestinalorganoid cells. FIG. 42A) enterocyte, tissue stained with Caudal TypeHomeobox 2 (CDX2) and Fatty Acid Binding Protein 2 (FABP2); FIG. 42B)Goblet cells, tissue stained with CDX2 and Mucin 2 (MUC2); FIG.42C)Paneth cells, tissue stained with CDX2 and lysozyme; and FIG. 42D)enteroendocrine cells, tissue stained with CDX2 and Chromatogranin A(parathyroid secretory protein 1), typically located in located insecretory vesicles.

FIG. 43A-C: Shows exemplary graphs demonstrating IFNgamma effects onhuman intestinal epithelial cells derived from IPSCs in microfluidicchips. Graphs show a loss of electrical resistance (TEER) and a loss ofconnections between epithelial cells treated with IFNgamma. FIG. 43A)TEER was reduced over time with IFNgamma treatment while control andTNFalpha treated cells showed increased TEER. FIG. 43B) FITC dextrinadded to the apical channel showed a similar loss as permeabilityco-efficients, and FIG. 43C) showed increased amounts of FITC dextrin inthe basal layer (after addition to the apical layer) for IFNgammatreated cells.

FIG. 44A-E: Shows Exemplary “Gut On A Chip” Technology. FIG. 44A) Showsschematic illustration of chip; FIG. 44B and FIG. 44C) shows photographswith overlays identifying parts and sizes of a “Gut On A Chip”; FIG.44C) additionally shows a micrograph of the membrane; FIG. 44D) Showsschematic illustration of a chip without and with mechanical strain withmicrographs of resulting cells below each representation; and FIG. 44E)shows a graph of substrate strain (%) vs. cell strain (%) in relation toapplied pressure (kPa).

FIG. 45A-C: Shows Epithelial Cells Growing in Channels of a “Gut On AChip”. Examples of seeded channels were fluorescently stained FIG. 45A)with DAPI (nuclei), FIG. 45B) E-cadherin, with an overlap of the twofluorescent channels shown in FIG. 45C).

FIG. 46: Shows exemplary cells cultured under static conditions for 6days in a microfluidic chip. Cells do not form a continuous layer.

FIG. 47: Shows exemplary cells cultured under flow conditions for 6 daysin a microfluidic chip. Cells form a continuous layer.

FIG. 48A-I: Shows graphs of relative expression of exemplary genemarkers between Caco-2 epithelial cells and intestinal enteroids grownin chips treated with IFNgamma. Expression was normalized to (GADPH),with and without IFNgamma treatment: FIG. 48A) IDO1 (indoleamine2,3-dioxygenase 1); FIG. 48B) GBP1 (guanylate binding protein 1); FIG.48C) GBP4 (guanylate binding protein 4); FIG. 48D) LYZ (Lysozyme); FIG.48E) PLA2G2A (Phospholipase A2 Group IIA); FIG. 48F) a secretedantibacterial lectin (RegIIIγ); FIG. 48G) LRG5 (Leucine Rich RepeatContaining G Protein-Coupled Receptor 5); FIG. 48H) OLM4 (Olfactomedin4); and FIG. 481) MUC4 (Mucin 4).

FIG. 49: Shows a representative image of how the chip looks after 12days. Twelve days after seeding chips, cells were confluent with acontinuous layer extending past the bend on the end of the upper channelof the chip.

FIG. 50: Shows a representative cross section cut along the axis of redline. A photographic view is shown in FIG. 51, with staining of cellsshown in the following figures.

FIG. 51: Shows an image of a cross section (viewing on end) ofmicrofluidic chip. A light micrograph of the cut axis through the chipshows the intestinal cells with microvilous-like structures growing onthe membrane in the upper channel of the chip. For reference, themembrane, lower channel, and vacuum chambers are identified in theimage.

FIG. 52: Presents an exemplary micrograph showing epithelial cellsderived from human intestinal organoids forming villous like structuresin response to a continuous flow of media in an upper and lower chamberof a small microfluidic device. Double staining shows Caudal TypeHomeobox 2 (CDX2) (red) and E-Cadherin (blue).

FIG. 53: Presents an exemplary micrograph showing stained epithelialcells and a cytoplasmic protein. Triple imminofluorsecence stainingshows the presence of Caudal Type Homeobox 2 (CDX2) (red) and E-Cadherin(blue) compared to Fatty Acid Binding Protein 2 (FABP2) (green).

FIG. 54: Presents an exemplary micrograph showing epithelial cellsderived from and a cytoplasmic protein. Triple imminofluorsecencestaining shows the presence of Caudal Type Homeobox 2 (CDX2) (red) andE-Cadherin (blue) compared to ZO-1 (green).

FIG. 55A-E: Shows exemplary images taken after seeding chips. FIG. 55A)7.5×106 cells/mL (300K in 40 uL); FIG. 55B) 6.25×106 cells/mL (250K in40 uL); FIG. 55C) 5.0×106 cells/mL (200K in 40 uL; FIG. 55D) 3.75×106cells/mL (150K in 40 uL); and FIG. 55E) 2.5×106 cells/mL (100K in 40uL).

FIG. 56: Shows exemplary magnified images of nonconfluent areas afterseeding chips. Enteroid cells seeded at 3.75×10⁶ cell/mL (150K in 40 uL)(compare to FIG. 55D). Red circle outlines a nonconfluent area.

FIG. 57: Shows exemplary magnified images of nonconfluent areas afterseeding chips with fewer cells than previous image. Enteroid cellsseeded at 2.5×10⁶ cell/mL (100K in 40 uL) (compare to FIG. 55E). Redcircles outline nonconfluent areas.

FIG. 58: Shows exemplary schematic Experimental Design for media testingon cell growth. In part, this design is to determine whether mediacontaining complete growth factors should be used in both upper-apicaland lower-basal channels for growing intestinal enteroid cells in themicrofluidic chip.

FIG. 59A-D: Shows exemplary Day 6 magnified images of intestinalenteroid cells growing on chips comparing media formulations in upper(apical) and lower (basal) channels. Media comparisons are: FIG. 59A)Complete(A)/Complete(B); FIG. 59B) GFR(A)/ Complete(B); FIG. 59C)Complete(A)/GFR(B); and FIG. 59D) GFR(A)/GFR(B).

FIG. 60A-C: Shows exemplary Day 7 magnified images of intestinalenteroid cells growing on chips comparing media formulations in upper(apical) and lower (basal) channels. Media comparisons are: FIG. 60A)Complete(A)/Complete(B); FIG. 60B) GFR(A)/ Complete(B); and FIG. 60C)Complete(A)/GFR(B).

FIG. 61A-B: Shows exemplary magnified images of intestinal enteroidcells growing on chips showing growth differences between two mediaformulations inducing microvillous-like structures. Media comparisonsare: FIG. 61A) Complete(A)/Complete(B) and FIG. 61B) GFR(A)/Complete(B).

FIG. 62A-F: Shows exemplary flow cytometry dot plots of enteroidiPS-derived intestinal cells as percentages of epithelial andnon-epithelial size gated cells from a microfluidic chip after 12 daysof incubation. FIG. 62A) Scatter plot showing intestinal cells sizegated as outlined at the flat end of the arrow into FIG. 62B) two-colorfluorescence dot plots showing background (auto) fluorescent intensityon two fluorescent channels and in *-fluorescent gated areas.Autofluorescence in gated areas for each fluorescent channel (*-outlinedfor fluorescent gating) shows 0.212% fluorescence (*-upper leftquadrant) and 0.004% (*-lower right quadrant) with a cell populationemitting autofluorescence on both channels shown in the populationgrouping in the lower left quadrant of the plot; FIG. 62C) Scatter plotshowing cells previously incubated with secondary fluorescent antibodyonly (another control for background) with cells gated as above for FIG.62D) two-color fluorescence dot plots for measuring backgroundfluorescence in high intensity areas for each channel (*-outlined forfluorescent gating) shows 0.149% fluorescence (*-upper left quadrant)and 0.00% (*-lower right quadrant); FIG. 62E) Cells fluorescentlystained with Epithelial Cell Adhesion Molecule (EpCAM) antibody (foridentifying epidermal cells), then gated for size as in A into atwo-color fluorescence dot plot, shows 83.4% EpCAM+ epithelial cells(*-outlined for fluorescent gating in upper left quadrant); and FIG.62F) Cells fluorescently stained with Vimentin, a type III intermediatefilament (IF) protein expressed in non-epithelial cells, then gated forsize as in A into a two-color fluorescence dot plot shows 15.6%Vimentin+non-epithelial cells (*-outlined for fluorescent gating inlower right quadrant).

FIG. 63A-D: Shows exemplary flow cytometry fluorescent dot plots of sizegated populations of enteroid iPS-derived intestinal cells that are notepithelial cells, from a microfluidic chip after 12 days of incubation.Cells were fluorescently stained with an antibody for identifying thefollowing cells as a percentage of the population gated intotwo-fluorescence plots: FIG. 63A) Paneth cells 5.03% (*-outlined in thelower right quadrant); FIG. 63B) Enteroendocrine cells 0.153%(*-outlined/fluorescently gated in the lower right quadrant); FIG. 63C)Goblet cells 0.131% (*-outlined/fluorescently gated in the lower rightquadrant); and FIG. 63D) Enterocytes 1.06% (*-outlined/fluorescentlygated in the lower right quadrant).

FIG. 64A-D: Shows exemplary flow cytometry fluorescent dot plots ofenteroid iPS-derived intestinal cells as percentages of epithelial andnonepithelial size gated cells from a microfluidic chip after 12 days ofincubation. Intestinal cell populations from size gated cells then gatedinto fluorescent intensity dot plots: FIG. 64A) Cells incubated with anisotype antibody control for the EpCAM primary antibody showing cellshaving 0.855% background fluorescence (*-outlined/gated in the upperleft quadrant); FIG. 64B) Cells incubated with secondary antibodywithout primary antibody having 0.065% background fluorescence(*-outlined/gated in the lower right quadrant); FIG. 64C) EpCAM+epithelial cells as 72% of the intestinal cell population; and FIG. 64D)Vimentin+ non-epithelial cells: 28.6% of the intestinal cell population.

FIG. 65A-C: Shows exemplary florescent micrographs of pulse-chasedmitotic/dividing cells in intestinal villi in a microfluidic chip. EdUlabeled (green) mitotic/dividing cells are shown in contrast toepithelial cells expressing E-cadherin (red) and nuclei stained withDAPI (blue). FIG. 65A) After a 4 hour pulse; then labeled cells areshown after FIG. 65B) a 72 hour chase and FIG. 65C) a 120 hour chase.

FIG. 66A-C: Shows exemplary florescent micrographs of pulse-chaseddividing cells located at the base of intestinal villi then moving intoupper villi structures growing in a microfluidic chip. EdU labeled(green) mitotic/dividing cells are shown in contrast to nuclei stainedwith DAPI (blue). EdU labeled (green) mitotic/dividing cells are locatedat the base of the intestinal microvilli FIG. 66A) after a 2 hour pulse;then labeled cells are located in villi structures after FIG. 66B) a 24hour chase and FIG. 66C) a 72 hour chase.

FIG. 67A-C: Shows exemplary florescent micrographs of pulse-chasedmitotic/dividing cells in intestinal villi in a microfluidic chip. EdUlabeled (green) mitotic/dividing cells are shown in contrast toepithelial cells expressing E-cadherin (red) and nuclei stained withDAPI (blue). EdU labeled (green) mitotic/dividing cells are located atthe base of the intestinal microvilli FIG. 67A) after a 2 hour pulse;then labeled cells are located in villi structures after FIG. 67B) a 24hour chase and FIG. 67C) a 72 hour chase.

FIG. 68A-C: Shows exemplary florescent micrographs of EdU labeledpulse-chased mitotic/dividing cells in intestinal villi in amicrofluidic chip as shown in FIG. 61. EdU labeled (green)mitotic/dividing cells are more clearly shown at the base of theintestinal microvilli without epithelial or nuclear stains FIG. 68A)after a 2 hour pulse; then labeled cells are located in villi structuresafter FIG. 68B) a 24 hour chase and FIG. 68C) a 72 hour chase.

FIG. 69A-B: Shows schematic diagrams of time line comparisons betweenintestinal enteroid cells derived from iPS cells. In one embodiment,cells are used FIG. 69A) directly or FIG. 69B) after freezing andthawing. Under both conditions, chips have epithelium containing villi(villous) structures.

FIG. 70: Shows a schematic diagram of a 3 organ circuit, wherein 3micofludic chips for 3 different organ-on-chips are fluidically attachedthrough basal channels. For reference, the upper-apical channel is shownin a solid line while the lower-basal channel is shown in a dotted line.

FIG. 71: Shows a schematic diagram of a 3 organ circuit, wherein 3micofludic chips for 3 different organ-on-chips are partiallyfluidically attached, i.e. through apical or basal channels.

FIG. 72: Shows a schematic diagram of a 2 organ circuit, wherein 2micofludic chips for 2 different organ-on-chips are partiallyfluidically attached, i.e. through the apical channels.

FIG. 73: Shows a schematic diagram of an exemplary anatomicalrelationship between embryonic foregut-midgut-hindgut regions and matureareas of the gastrointestinal system. An arrow points to an exemplaryAntrum/pyloric region in the stomach.

DESCRIPTION OF ENDOCRINE DISRUPTING CHEMICALS (EDCs)

Persistent human exposure to elevated levels of man-made endocrinedisrupting chemicals (EDCs) during critical periods in fetal developmentmay lead to long-term disruption of metabolic homeostasis in endocrinetissue progenitors, thus contributing to childhood obesity. A feasibleplatform to test EDC-induced developmental abnormalities in human gutand brain endocrine tissues does not exist. Thus, the Inventorsdeveloped a platform to determine the effect of low-dose chronicexposure to common EDCs that contaminate the Inventors' food and watersupply including, perfluorooctanoic acid (PFOA), tributyltin (TBT) andbutyl hydroxytoluene (BHT), using two human induced pluripotent stemcell (hiPSC)-derived endocrine tissues—developing foregut epitheliumcells (iFGEs) and neuropeptidergic hypothalamic neurons (iHTNs).

As described, endocrine disrupting chemicals (EDCs) are a group ofpervasive environmental obesogens that have been shown to play adisruptive role in normal tissue development by targeting hormonalsignaling pathways and hormonal control of hunger and satiety. Obesogensmay also alter basal metabolic rate, by shifting energy balance in favorof calorie storage, thereby contributing to obesogenic phenotypes.

The greater risk lies in the fact that these EDCs can betransgenerationally exposed from the mother to the offspring in uterowhich can bring about effects such as epigenetic imprinting via repeatedexposure during critical windows of stem cell development e.g.predisposes mesenchymal stem cells to preferentially differentiate intoadipocytes Besides, EDCs transmitted across generations have been shownto have an adverse impact for at least three generations of mice.Although not many human studies show a direct link between obesogens anddevelopmental defects, there is epidemiological evidence thatenvironmental chemicals have detrimental effects in early developmentand may have life-long effects on the physiology of the offspring. Thisis also a transgenerational phenomenon whereby effects can be seen evenin the subsequent generations. Further, increased body mass index andobesity is transmitted across generations as a result of maternalobesity during gestation. Taken together, the environmental chemicalsand their impact in human stem needs to be addressed urgently with ahuman-specific developmental screening platform. Ubiquitous “obesogenic”endocrine disrupting chemicals (EDCs) are discussed below in some of theexamples. EDCs include but are not limited to like phthalateplasticizers, organotins, perfluorochemicals, and food additives.Exposure is mainly through human food during critical windows of stemcell development in utero or early-life.

A. Compound Screening.

Described herein are the effects of 3 different EDCs individually and incombination—perfluorooctanoic acid (PFOA), tributyltin (TBT) andbutylhydroxy toluene (BHT). PFOA is known to be surfactant influoropolymers and is known to persist indefinitely in the environment.According to a study in 2007, about 98% of the US population hasdetectable levels of PFOA in their blood that can expose itself viaindustrial waste, stain resistant carpets, house dust, water andcookware coating. TBT, an organotin, is used as an anti-fouling agentused in paints to keep ships from bio-fouling. However, its presence inhouse dust is a major source of human exposure. BHT is a common foodadditive, personal care and cosmetic product ingredient, pesticide,plastic and rubber ingredient. It is however also utilized as anantioxidant in commonly consumed breakfast cereal brands. The use ofhuman induced pluripotent stem cells (hiPSCs) to elucidate the adverseeffects and mechanisms of chronic low-dose EDC exposures on developinggut and hypothalamic neuropeptidergic neurons, and serves as a platformfor mimicking the in utero exposure to EDCs.

Described herein is a method of compound screening, including providinga quantity of differentiated induced pluripotent stem cells (iPSCs),contacting the differentiated iPSCs with one or more compounds,measuring one or more properties of the differentiated iPSCs, whereinmeasurement of the one or more properties of the differentiated iPSCsidentifies characteristics of the one or more compounds. In variousembodiments, compound screening comprises screening for endocrinedisruption. In various embodiments, characteristics of the one or morecompounds comprise inducing phorphorylation of NF-kB. In variousembodiments, characteristics of the one or compounds comprise decreasein mitochondrial respiration. In various embodiments, characteristics ofthe one or compounds comprise decrease in expression of one or more ofSCO2, POLRMT, TFAM and CYTB5. In various embodiments, the differentiatediPSCs are foregut epithelium. In various embodiments, the differentiatediPSCs are hypothalamic neurons.

B. Differentiating Induced Pluripotent Stem Cells (iPSC).

Further described herein is a method of differentiating inducedpluripotent stem cells, including providing a quantity of inducedpluripotent stem cells (iPSCs), and culturing in the presence of one ormore factors, wherein the one or more factors are capable ofdifferentiating the iPSCs.

In various embodiments, the iPSCs are differentiated into definitiveendoderm by culturing in the presence of one or more factors comprisingActivin A and Wnt3A. In various embodiments, culturing in the presenceof one or more factors comprising Activin A and Wnt3A is for about 3days. In various embodiments, the differentiated iPSCs are initiallycultured under serum-free conditions, followed by addition of serum. Invarious embodiments, definitive endoderm is differentiated into foregutspheroids by further culturing in the presence of one or more factorscomprising CHIR99021, FGF (FGF4), LDN, and Retinoic Acid (RA). Invarious embodiments, culturing in the presence of one or more factorscomprising CHIR99021, FGF (FGF4), LDN, and Retinoic Acid (RA) is forabout 3 days. In various embodiments, foregut spheroid is differentiatedinto foregut epithelium by culturing a coated surface. In variousembodiments, foregut spheroid is differentiated into foregut epitheliumby additional culturing in the presence of one or more factors epidermalgrowth factor (EGF). In various embodiments, additional culturing in thepresence of one or more factors comprising epidermal growth factor (EGF)is for about 20 days. In various embodiments, the differentiated iPSCsare foregut epithelium. In various embodiments, the foregut epitheliumexpresses one or more of SOX2, SOX17, PDX1, GKN1, PGA5, TAS1R3 and TFF2.In various embodiments, the foregut epithelium expresses one or more ofsynaptophysin (SYP), somatostatin, serotonin, gastrin, ghrelin andpeptide YY. In various embodiments, the foregut epithelium does notexpress Caudal Type Homeobox 2 (CDX2).

In various embodiments, the iPSCs are initially cultured in the presenceof ROCK-inhibitor Y27632. In various embodiments, the iPSCs aredifferentiated into neuroectoderm by culturing in the presence of one ormore factors comprising LDN193189 and SB431542. In various embodiments,culturing in the presence of one or more factors comprising LDN193189and SB431542 is for about 2 days. In various embodiments, theneuroectoderm is differentiated into ventral diencephalon by culturingin the presence of one or more factors comprising moothened agonist SAG,purmorphamine (PMN) and IWR-endo. In various embodiments, culturing inthe presence of one or more factors comprising moothened agonist SAG,purmorphamine (PMN) and IWR-endo is for about 5-6 days. In variousembodiments, ventral diencephalon is matured by culturing in thepresence of one or more factors comprising DAPT, retinoic acid (RA). Invarious embodiments, culturing in the presence of one or more factorscomprising DAPT, retinoic acid (RA) is for about 4-5 days. In variousembodiments, the mature ventral diencephalon is further matured byculturing in the presence of one or more factors comprising BDNF. Invarious embodiments, culturing in the presence of one or more factorscomprising BDNF is for about 20-27 days. In various embodiments, thedifferentiated iPSCs are hypothalamic neurons. In various embodiments,the hypothalamic neurons express one or more of AgRP (Agouti-relatedPeptide), MC4R (Melanocortin 4 receptor), Nkx2.1, NPY (Neuropeptide Y),and PCSK2 (Proprotein Convertase Subtilisin/Kexin Type 2).

Description of Intestinal Cells and Microfluidic Chips

In one embodiment, the present invention contemplates a method ofculturing cells, comprising: a) providing a fluidic device comprising amembrane, said membrane comprising a top surface and a bottom surface;b) seeding cells on said bottom surface; and c) culturing said seededcells under conditions that support the growth of an intestinalorganoid. In one embodiment, the cells are derived from an intestinaltissue biopsy sample of a patient diagnosed with a disorder of thegastrointestinal system. In one embodiment, the cells are derived frominduced pluripotent stem cells derived from a patient diagnosed with adisorder of the gastrointestinal system. In one embodiment, the patientis a human patient. In one embodiment, the gastrointestinal disorder isirritable bowel disease. In one embodiment, the method further comprisesseeding said cells on said top surface and culturing said top surfaceseeded cells under conditions that support the maturation of at leastone intestinal villa structure. In one embodiment, the at least oneintestinal villa structure is polarized toward an intestinal organoidlumen. In one embodiment, the at least one intestinal villa ismorphologically similar to an in vivo intestinal villa. In oneembodiment, the intestinal villa comprises an intestinal cell typeincluding, but not limited to, Paneth cells, goblet cells,enteroendocrine cells and enterocyte cells. In one embodiment, theintestinal cell type is confirmed by immunocytochemistry. In oneembodiment, the intestinal cell type comprises Igr5+. In one embodiment,the Paneth cells secrete antimicrobials. In one embodiment, the methodfurther comprises administering IFNgamma to the intestinal organoidunder conditions such that STAT1 is phosphorylated. In one embodiment,the method further comprises administering IFNgamma to the intestinalorganoid under conditions such that an IFNgamma responsive gene isupregulated. In one embodiment, the IFNgamma responsive gene includes,but is not limited to, IDO1, GBP4 and/or GBP5. In one embodiment, theIFNgamma administration further upregulates intestinal epithelialsubtype-specific genes. In one embodiment, the intestinal epithelialsubtype-specific genes include, but are not limited to, phospholipase A2group 2A and/or Muc4. In one embodiment, the method further comprisesmeasuring gene expression in said intestinal organoid. In oneembodiment, the method further comprises measuring antimicrobialsecretion in said intestinal organoid. In one embodiment, the methodfurther comprises assessing the influence of an agent including, but notlimited to, luminal microbes, immune cells and/or cyokines on intestinalorganoid function.

In one embodiment, the present invention contemplates a gut-intestinalchip where at least one population of cells is derived from a patientdiagnosed with a disorder of the gastrointestinal system. While it isnot intended that the present invention be limited to a particulargastrointestinal disorder, in one embodiment, the disorder is irritablebowel disease (IBD). Although it is not necessary to understand themechanism of an invention it is believed that a gut-intestinal chipmodel may facilitate understanding of the role of the intestinalepithelium in IBD by combining microfluidic technology and IPSC-derivedhuman intestinal organoids.

Inflammatory bowel disease (IBD) is believed to be a complex polygenicdisorder that may be characterized by recurrent mucosal injury. It isbelieved to be caused by a dysregulated immune response to luminalmicrobes in genetically susceptible individuals. While numerous lines ofevidence suggest that the intestinal epithelium may also play a role,it's precise role in IBD has remained elusive due a lack of suitable invitro models.

The development of intestinal organoid technology achieved advances inthis area, whereby human intestinal organoids (HIOs) from controlindividuals/IBD patients could be generated from induced pluripotentstem cells (iPSCs) or biopsy samples. However, in the context of IBD,this technology is very challenging to use. Given that HIOs arepolarized towards the lumen, studies examining intestinal permeabilityor bacterial-epithelial interactions are facilitated by providing accessthe interior of the HIOs which is laborious and requires specialistequipment. In addition, studies examining epithelial-immune cellinteractions are hampered as HIOs are embedded in a matrix.

One advantage of some embodiments of the present invention overcome suchlimitations by providing a gut-on-a-chip technology. In one embodiment,iPSCs were directed to form HIOs and were subsequently dissociated to asingle cell suspension. These cells were then seeded into a smallmicrofluidic device (SMD) which is composed of two chambers separated bya porous flexible membrane. A continuous flow of media in both the upperand lower chamber of the device resulted in the spontaneous formation ofpolarized villous-like structures that are similar to those found invivo. The presence of Paneth cells, goblet cells, enteroendocrine cellsand enterocytes in these structures was confirmed by immunocytochemistrywhile in situ hybridization revealed the presence of lgr5+ cells.Secretion of antimicrobials from Paneth cells was detected by ELISA andadministration of IFNgamma to the lower channel resulted in thephosphorylation of STAT1 and significant upregulation of IFNgammaresponsive genes including, but not limited to, IDO1, GBP4 and/or GBP5.Interestingly, phospholipase A2 group 2A and Muc4, two genes specific tointestinal epithelial subtypes, were also upregulated. When compared toCaco2 cells, there was no corresponding upregulation of genes associatedwith these epithelial subtypes.

In one embodiment, the present invention contemplates a system wherebyiPSC-derived intestinal epithelium can be incorporated into SMDs andchanges in gene expression and antimicrobial secretion can be measured.Previous demonstration of HIO generation from lymphoblastoid cell lines(LCLs), predicts that genotyped IBD-LCLs stored by the NIDDK can beobtained to generate intestinal epithelium containing genetic variantsassociated with IBD. Although it is not necessary to understand themechanism of an invention, it is believed that a gut-on-a-chiptechnology allows an assessment as to how these variants influence thefunctioning of gut tissue and response to various luminal microbesand/or immune cells/cytokines.

Described herein is a microfluidic device using induced pluripotent stemcell (iPSC) derived intestinal epithelium. The device permits the flowof media resulting in successful villi formation and peristalsis.Importantly, the use of iPSC-derived epithelium allows for generation ofmaterial derived from IBD patients, thereby presenting an opportunityfor recapitulating genetic disease elements. Moreover, the use of iPSCsas source material further allows production of other cell types, suchas immune cells, which can be studied in parallel to further investigatetheir contribution to disease progression.

As described, organs-on-chips are microfluidic devices for culturingcells in continuously perfused, micrometer sized chambers. Thecombination of artificial construction and living materials allowsmodeling of physiological functions of tissues and organs.

Microfluidic culture systems are often made by ‘soft lithography’, ameans of replicating patterns etched into silicon chips in morebiocompatible and flexible materials. A liquid polymer, such aspoly-dimethylsiloxane (PDMS), is poured on an etched silicon substrateand allowing it to polymerize into an optically clear, rubber-likematerial. This allows one to specify the shape, position and function ofcells cultured on chips. Alternatively, inverting the PDMS mold andconformally sealing it to a flat smooth substrate, allows creation ofopen cavities in the such as linear, hollow chambers, or ‘microfluidicchannels’ for perfusion of fluids. Such PDMS culture systems areoptically clear, allowing for high-resolution optical imaging ofcellular responses. In some instances, miniaturized perfusionbioreactors for culturing cells are made by coating the surface ofchannels with extracellular matrix (ECM) molecules. Cells can introducedvia flow through the channel for capture and adherence to the ECMsubstrate. Additional details are found in Bhatia and Ingber,“Microfluidic organs-on-chips.” Nat Biotechnol. (2014) 8:760-72, whichis fully incorporated by reference herein.

Importantly, microfluidic chips provide control over system parametersin a manner not otherwise available in 3D static cultures orbioreactors. This allows study of a broad array of physiologicalphenomena. In some instances, integration of microsensors allows studyof cultured cells in the microenvironmental conditions. Further, flowcontrol of fluid in chips allows the generation of physical and chemicalgradients, which can be exploited for study of cell migration, analysisof subcellular structure and cell-cell junctional integrity. In additionto detection and control of such mechanical forces, control of cellpatterning allows study of physiological organization and interaction.For example, different cell types can be plated in distinct physicalspaces, and using the above described techniques, shaped by micromoldingtechniques into organ-like forms, such as the villus shape of theintestine. Chips also allow the complex mechanical microenvironment ofliving tissues to be recapitulated in vitro. Cyclical mechanical straincan be introduced using flexible side chambers, with continuous rhythmicstretching relaxing lateral walls and attached central membranes. Thiscyclic mechanical deformation and fluid shear stresses introduced inparallel, mimic cellular exposure in living organs, including intestinalfunction such as peristalsis.

In the context of investigating intestinal disease, human intestinalepithelial cells (Caco-2) have been cultured in the presence ofphysiologically relevant luminal flow and mimicking peristalsis-likemechanical deformations. Caco-2 cells can be cultured on a flexible,porous ECM-coated membrane within a microfluidic device exposed both totrickling flow. Analogous to that in the gut lumen, and to cyclicmechanical distortion, these mechanical forces mimic peristalsis-likemotions of the living intestine, and interestingly, promotereorganization into 3D undulating tissue structures lined by columnarepithelial cells that resemble the architecture of the villus of thesmall intestine. Relevant specialized features include reestablishmentof functional basal proliferative cell crypts, differentiation of allfour cell lineages of the small intestine types (absorptive,mucus-secretory, enteroendocrine and Paneth), secretion of high levelsof mucin and formation of a higher resistance epithelial barrier.

Importantly, fluid flow allows culturing the human intestinal cells withliving commensal bacteria in the lumen of the gut-on-a-chip withoutcompromising cell viability. In static formats, intestinal cellscultured in the presence of bacteria cannot survive based on bacterialovergrowth. However, continuous flow allows for sustained exposure ofbacteria for extended periods of time while maintaining cellularviability. This approach opens entirely new avenues for microbiomeresearch. Additional details are found in Kim et al., “Contributions ofmicrobiome and mechanical deformation to intestinal bacterial overgrowthand inflammation in a human gut-on-a-chip.” Proc Natl Acad Sci USA.(2016) 113:E7-E15.

Most studies with organs-on-chips have been carried out on establishedcell lines or primary cells. Of great interest is applying themethodologies and designs to stem cells, and particularly inducedpluripotent stem cells (iPSCs). In particular, use of patient-derived,including disease-specific cells allows potential to model diseasedorgans. In the context of intestinal disease, the use of iPSCs derivedfrom IBD patients allows study of an entire repertoire of geneticvariations associated with IBD, not otherwise if limited to using cellssuch as Caco-2. Moreover, iPSCs as a cell source allow production of notonly the intestinal cells of interest, but also corresponding immunecells (e.g., macrophages, neutrophils, and dendritic cells) from thesame individual/IBD patient, to investigate potential influence indisease pathology.

Described herein is a microfluidic device using induced pluripotent stemcell (iPSC) derived intestinal epithelium. The device permits the flowof media resulting in successful villi formation and peristalsis.Importantly, the use of iPSC-derived epithelium allows for generation ofmaterial derived from IBD patients, thereby presenting an opportunityfor recapitulating genetic disease elements. Moreover, the use of iPSCsas source material further allows production of other cell types, suchas immune cells, which can be studied in parallel to further investigatetheir contribution to disease progression. The purpose of this inventionis to ultimately understand how the intestinal epithelium is influencedby genetics, other immune cell types and environmental stimuli such asinflammatory cytokines and bacteria.

A. Microfluidic Device With Intestinal Cells.

Described herein are methods for manufacturing a microfluidic deviceincluding a population of intestinal cells. In various embodiments, themethod includes generation of human intestinal organoids (HIOs) frominduced pluripotent stem cells (iPSCs), and seeding of intestinalepithelial cells into the microfluidic device. In various embodiments,the microfluidic apparatus including a population of intestinal cellswith an organized structure, including disaggregating HIOs into singlecells and adding the single cells to the apparatus. In variousembodiments, the single cells are purified based on CD326+ expressionbefore addition to the apparatus. In various embodiments, adding thesingle cells to the apparatus includes resuspension in a media includingone or more of: ROCK inhibitor, SB202190 and A83-01. In variousembodiments, the HIOs are cultured in the presence of ROCK inhibitorprior to disaggregation. In various embodiments, the HIOs are derivedfrom iPSCs. In various embodiments, the iPSCs are reprogrammedlymphoblastoid B-cell derived induced pluripotent stem cells(LCL-iPSCs). In various embodiments, the iPSCs are reprogrammed cellsobtained from a subject afflicted with an inflammatory bowel diseaseand/or condition. In various embodiments, derivation of HIOs from iPSCsincludes generation of definitive endoderm by culturing iPSCs in thepresence of Activin A and Wnt3A, differentiation into hindgut byculturing definitive endoderm in the presence of FGF and either Wnt3A orCHIR99021, collection of epithelial spheres or epithelial tubes,suspension of epithelial spheres or epithelial tubes in Matrigel, andculturing in the presence of CHIR99021, noggin and EGF. In variousembodiments, the organized structure includes villi. In variousembodiments, the villi are lined by one or more epithelial cell lineagesselected from the group consisting of: absorptive, goblet,enteroendocrine, and Paneth cells. In various embodiments, the organizedstructure possesses barrier function, cytochrome P450 activity, and/orapical mucus secretion.

B. Generation Of Human Intestinal Organoids (HIOs) From iPSCs.

In various embodiments, the method includes generation of HIOs fromiPSCs, including differentiation of iPSCs into definitive endoderm,epithelial structures and organoids. In various embodiments, inductionof definitive endoderm includes culturing of iPSCs with Activin A andWnt3A, for 1, 2, 3, 4 or more days, and increasing concentrations of FBSover time. In various embodiments, induction of definitive endodermincludes culturing of iPSCs with Activin A (e.g., 100 ng/ml), Wnt3A (25ng/ml), for 1, 2, 3, 4 or more days, and increasing concentrations ofFBS over time (0%, 0.2% and 2% on days 1, 2 and 3 respectively). Forexample, induction of definitive endoderm includes culturing of iPSCswith Activin A (e.g., 100 ng/ml), Wnt3A (25 ng/ml), for 1, 2, 3, 4 ormore days, and increasing concentrations of FBS over time (0%, 0.2% and2% on days 1, 2 and 3 respectively). In various embodiments, theconcentration of Activin A includes about 0-25 ng/ml, about 25-50 ng/ml,about 50-75 ng/ml, about 100-125ng/ml, about 125-150 ng/ml. In variousembodiments, the concentration of Wnt3A includes about −25 ng/ml, about25-50 ng/ml, about 50-75 ng/ml, about 100-125ng/ml, about 125-150 ng/ml.In various embodiments, the concentrations of FBS over time includeabout 0%-0.2%, about 0.2%-0.5%, about 0.5%-1%, about 1%-2%, and 2% ormore on each of days 1, 2 and 3 respectively. In various embodiments,formation of hindgut includes culturing of definitive endoderm cells for1, 2, 3, 4 or more days in media such as Advanced DMEM/F12 with FBS andFGF4. In various embodiments, formation of hindgut includes culturing ofdefinitive endoderm cells for 1, 2, 3, 4 or more days in media includeFBS at a concentration of 0%-0.2%, about 0.2%-0.5%, about 0.5%-1%, about1%-2%, and 2% or more and concentration of FGF4 at about 50-100 ng/ml,about 100-250 ng/ml, about 250-500ng/ml, and 500 ng/ml or more. Forexample, formation of hindgut can include culturing of definitiveendoderm cells for 1, 2, 3, 4 or more days in media such as AdvancedDMEM/F12 with 2% FBS and FGF4 (500 ng/ml). In various embodiments,Wnt3A, CHIR99021 or both are added. In various embodiments, theconcentration of Wnt3A includes about 100-250 ng/ml, about 250-500ng/ml,and 500 ng/ml, the concentration of CHIR99021 is about 0.5-1 μM, about1-1.5 μM , about 1.5-2 μM or 2 μM or more are added. For example, bothWnt3A (500 ng/ml), CHIR99021 (2 μM) or both are added. In variousembodiments, after about 3-4 days, the method includes isolation oforganoids including free floating epithelial spheres and looselyattached epithelial tubes. In various embodiments, the isolatedorganoids are suspended in Matrigel and then overlaid in intestinalmedium containing CHIR99021, noggin, EGF and B27. In variousembodiments, the isolated organoids are suspended in Matrigel and thenoverlaid in intestinal medium containing CHIR99021, noggin, EGF and B27.In various embodiments, the concentration of CHIR99021 is about 0.5-1μM, about 1-1.5 μM , about 1.5-2 μM or 2 μM, the concentration of nogginat about 50-100 ng/ml, about 100-250 ng/ml is about 250-500ng/ml, and500 ng/ml or more, the concentration of EGF at about 50-100 ng/ml, about100-250 ng/ml, about 250-500 ng/ml, and 500 ng/ml or more and theconcentration of B27 is about 0.25×-0.5×, about 0.5-1×, about 1×-2× or2× or more. For example, the media contains CHIR99021 (2 μM), noggin(100 ng/ml) and EGF (100 ng/ml) and B27 (1×). In various embodiments,HIOs are passaged every 7-10 days thereafter. In various embodiments,the population of intestinal are an organized population includingfeatures of intestinal organs. In various embodiments, the inestitinalcells are organized into villi. In various embodiments, the villi arelined by all four epithelial cell lineages of the small intestine(absorptive, goblet, enteroendocrine, and Paneth). In variousembodiments, the population of intestinal cells possess barrierfunction, drug-metabolizing cytochrome P450 activity, and/or apicalmucus secretion.

C. Intestinal Cell Populations Includes An Organized Structure.

Described herein is a microfluidic apparatus including a population ofintestinal cells, wherein the population includes an organizedstructure. In various embodiments, the organized structure includesvilli. In various embodiments, the villi are lined by one or moreepithelial cell lineages selected from the group consisting of:absorptive, goblet, enteroendocrine, and Paneth cells. In variousembodiments, the organized structure possesses barrier function,cytochrome P450 activity, and/or apical mucus secretion. In variousembodiments, the intestinal cells are derived from human intestinalorganoids (HIOs) disaggregated into single cells and purified based onCD326+ expression. In various embodiments, the HIOs are derived fromiPSCs by a method including generation of definitive endoderm byculturing iPSCs in the presence of Activin A and Wnt3A, differentiationinto hindgut by culturing definitive endoderm in the presence of FGF andeither Wnt3A or CHIR99021, collection of epithelial spheres orepithelial tubes, suspension of epithelial spheres or epithelial tubesin Matrigel, and culturing in the presence of CHIR99021, noggin and EGF.

Description of Generating Induced Pluripotent Stem Cells (iPSC)

The following are embodiments of methods relating to generating inducedpluripotent stem cells (iPSCs) from a somatic cell source, including butnot limited to white blood cells, in section A with an exemple of suchuse for generating iPSCs from an exemplary white blood cell source inthe form of lymphoblastoid B-cells in section B. Lymphoblastoid B-cellsare a type of white blood cell desirable for use as original sourcematerial to make iPSCs, subsequently reprogrammed via the methoddescribed herein, including in Secetion A below. These white blood cellderived iPSCs are later differentiated into other cell types, includingbut not limited to intestinal cells, hypothalamic neurons, endothelial,etc. Thus, the techniques for manipulation of the source materials, suchas described in Section A below and herein, using exemplary sourcematerials described in B below, and herein, are broadly capable ofgenerating the various differentiated cells described for use withmicrofluidic chips described herein.

A. Generating Induced Pluripotent Stem Cells (iPSC) From Somatic CellSources.

Also described herein is an efficient method for generating inducedpluripotent stem cells, including providing a quantity of cells,delivering a quantity of reprogramming factors into the cells, culturingthe cells in a reprogramming media for at least 4 days, whereindelivering the reprogramming factors, and culturing generates inducedpluripotent stem cells. In certain embodiments, the cells are primaryculture cells. In other embodiments, the cells are blood cells (BCs). Incertain embodiments, the blood cells are T-cells. In other embodiments,the blood cells are non-T-cells. In other embodiments, the cells aremononuclear cells (MNCs), including for example peripheral bloodmononuclear cells (PBMCs). In other embodiments, the cells are primarygranulocytes, monocytes and B-lymphocytes.

In certain embodiments, the reprogramming factors are Oct-4, Sox-2,Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and short hairpinRNAs targeting p53 (“shRNA-p53”). In other embodiments, thesereprogramming factors are encoded in a combination of vectors includingpEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL andpCXWB-EBNA1. This includes, for example, using about 0.5-1.0 ugpCXLE-hOCT3/4-shp53, 0.5-1.0 ug pCXLE-hSK, 0.5-1.0 ug pCXLE-UL, about0.25-0.75 ug pCXWB-EBNA1 and 0.5-1.0 ug pEP4 E02S ET2K. This includes,for example, using 0.83 ug pCXLE-hOCT3/4-shp53, 0.83 ug pCXLE-hSK, 0.83ug pCXLE-UL, 0.5 ug pCXWB-EBNA1 and 0.83 ug pEP4 E02S ET2K, wherein thestoichiometric ratio of SV40LT (encoded in pEP4 E02S ET2K) and EBNA-1(encoded in pCXWB-EBNA1) supports the reprogramming of non-T cellcomponent of blood, including peripheral blood mononuclear cells. Invarious embodiments, the reprogramming media is embryonic stem cell(ESC) media. In various embodiments, the reprogramming media includesbFGF. In various embodiments, the reprogramming media is E7 media. Invarious embodiments, the reprogramming E7 media includes L-AscorbicAcid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/orbFGF. In different embodiments, the reprogramming media comprises atleast one small chemical induction molecule. In certain otherembodiments, the reprogramming media includes PD0325901, CHIR99021,HA-100, and A-83-01. In other embodiments, the culturing the blood cellsin a reprogramming media is for 4-30 days.

In various embodiments, the BC-iPSCs are capable of serial passaging asa cell line. In various embodiments, the BC-iPSCs possess genomicstability. Genomic stability can be ascertained by various techniquesknown in the art. For example, G-band karyotyping can identify abnormalcells lacking genomic stability, wherein abnoinial cells possess about10% or more mosaicism, or one or more balanced translocations of greaterthan about 5, 6, 7, 8, 9, 10 or more Mb. Alternatively, genomicstability can be measured using comparative genomic hybridization (aCGH)microarray, comparing for example, BC-iPSCs against iPSCs from anon-blood cell source such as fibroblasts. Genomic stability can includecopy number variants (CNVs), duplications/deletions, and unbalancedtranslocations. In various embodiments, BC-iPSCs exhibit no more thanabout 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 Mbaverage size of amplification and deletion. In various embodiments,BC-iPSCs exhibit no more than about 20-30 Mb average size ofamplification and deletion. In various embodiments, BC-iPSCs exhibit nomore than about 30-40 Mb average size of amplification and deletion. Invarious embodiments, BC-iPSCs exhibit no more than about 40-50 Mbaverage size of amplification and deletion. In various embodiments, theaverage number of acquired de novo amplification and deletions inBC-iPSCs is less than about 5, 4, 3, 2, or 1. For example, de novoamplification and deletions in fib-iPSCs are at least two-fold greaterthan in PBMC-iPSCs. In various embodiments, the methods produces iPSCcell lines collectively exhibiting about 20%, 15%, 10%, 5% or lessabnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or morepassages when serially passaged as a cell line.

In different embodiments, reprogramming factors can also include one ormore of following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV4OLT,shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or a Mir-290 clustermicroRNA such as miR-291-3p, miR-294 or miR-295. In differentembodiments, the reprogramming factors are encoded by a vector. Indifferent embodiments, the vector can be, for example, a non-integratingepisomal vector, minicircle vector, plasmid, retrovirus (integrating andnon-integrating) and/or other genetic elements known to one of ordinaryskill. In different embodiments, the reprogramming factors are encodedby one or more oriP/EBNA1 derived vectors. In different embodiments, thevector encodes one or more reprogramming factors, and combinations ofvectors can be used together to deliver one or more of Oct-4, Sox-2,Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1,Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR-294 ormiR-295. For example, oriP/EBNA1 is an episomal vector that can encode avector combination of multiple reprogramming factors, such as pCXLE-hUL,pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, pEP4 EO2S T2K and pCXWB-EBNA1.

In other embodiments, the reprogramming factors are delivered bytechniques known in the art, such as nuclefection, transfection,transduction, electrofusion, electroporation, microinjection, cellfusion, among others. In other embodiments, the reprogramming factorsare provided as RNA, linear DNA, peptides or proteins, or a cellularextract of a pluripotent stem cell. In certain embodiments, the cellsare treated with sodium butyrate prior to delivery of the reprogrammingfactors. In other embodiments, the cells are incubated or 1, 2, 3, 4, ormore days on a tissue culture surface before further culturing. This caninclude, for example, incubation on a Matrigel coated tissue culturesurface. In other embodiments, the reprogramming conditions includeapplication of norm-oxygen conditions, such as 5% O₂, which is less thanatmospheric 21% O₂.

In various embodiments, the reprogramming media is embryonic stem cell(ESC) media. In various embodiments, the reprogramming media includesbFGF. In various embodiments, the reprogramming media is E7 media. Invarious embodiments, the reprogramming E7 media includes L-AscorbicAcid, Transferrin, Sodium Bicarbonate, Insulin, Sodium Selenite and/orbFGF. In different embodiments, the reprogramming media comprises atleast one small chemical induction molecule. In different embodiments,the at least one small chemical induction molecule comprises PD0325901,CHIR99021, HA-100, A-83-01, valproic acid (VPA), SB431542, Y-27632 orthiazovivin (“Tzv”). In different embodiments, culturing the BCs in areprogramming media is for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30days.

Efficiency of reprogramming is readily ascertained by one of manytechniques readily understood by one of ordinary skill. For example,efficiency can be described by the ratio between the number of donorcells receiving the full set of reprogramming factors and the number ofreprogrammed colonies generated. Measuring the number donor cellsreceiving reprogramming factors can be measured directly, when areporter gene such as GFP is included in a vector encoding areprogramming factor. Alternatively, indirect measurement of deliveryefficiency can be provided by transfecting a vector encoding a reportergene as a proxy to gauge delivery efficiency in paired samplesdelivering reprogramming factor vectors. Further, the number ofreprogrammed colonies generated can be measured by, for example,observing the appearance of one or more embryonic stem cell-likepluripotency characteristics such as alkaline phosphatase (AP)-positiveclones, colonies with endogenous expression of transcription factors Octor Nanog, or antibody staining of surface markers such as Tra-1-60. Inanother example, efficiency can be described by the kinetics of inducedpluripotent stem cell generation. For example, efficiency can includeproducing cell lines of normal karyotype, including the method producingiPSC cell lines collectively exhibiting about 20%, 15%, 10%, 5% or lessabnormal karyotypes over 4-8, 9-13, 13-17, 17-21, 21-25, or 29 or morepassages when serially passaged as a cell line.

B. Generating Lymphoblastoid B-Cell Derived Induced Pluripotent StemCells (“LCL-iPSCs”).

“LCL-iPSCs” are generated using techniques described in Section A above.

Described herein is a composition of lymphoblastoid B-cell derivedinduced pluripotent stem cells (“LCL-iPSCs”). In certain embodiments,the composition of B-cell derived induced pluripotent stem cellsincludes cells generated by providing a quantity of lymphoid cells(LCs), delivering a quantity of reprogramming factors into the LCs,culturing the LCs in a reprogramming media for at least 7 days, andfurther culturing the LCs in an induction media for at least 10 days,wherein delivering the reprogramming factors, culturing and furtherculturing generates the lymphoid-cell derived induced pluripotent stemcells. In certain embodiments, the reprogramming factors are Oct-4,Sox-2, Klf-4, c-Myc, Lin-28, SV40 Large T Antigen (“SV40LT”), and shorthairpin RNAs targeting p53 (“shRNA-p53”). In other embodiments, thesereprogramming factors are encoded in a combination of vectors includingpEP4 E02S ET2K, pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL. Incertain other embodiments, the reprogramming media includes PD0325901,CHIR99021, HA-100, and A-83-01. In other embodiments, the culturing theLCs in a reprogramming media is for 8-14 days and further culturing theLCs in an induction media is for 1-12 days.

In different embodiments, reprogramming factors can also include one ormore of following: Oct-4, Sox-2, Klf-4, c-Myc, Lin-28, SV40LT,shRNA-p53, nanog, Sall4, Fbx-15, Utf-1, Tert, or a Mir-290 clustermicroRNA such as miR-291-3p, miR-294 or miR-295. In differentembodiments, the reprogramming factors are encoded by a vector. Indifferent embodiments, the vector can be, for example, a non-integratingepisomal vector, minicircle vector, plasmid, retrovirus (integrating andnon-integrating) and/or other genetic elements known to one of ordinaryskill. In different embodiments, the reprogramming factors are encodedby one or more oriP/EBNA1 derived vectors. In different embodiments, thevector encodes one or more reprogramming factors, and combinations ofvectors can be used together to deliver one or more of Oct-4, Sox-2,Klf-4, c-Myc, Lin-28, SV40LT, shRNA-p53, nanog, Sall4, Fbx-15, Utf-1,Tert, or a Mir-290 cluster microRNA such as miR-291-3p, miR-294 ormiR-295. For example, oriP/EBNA1 is an episomal vector that can encode avector combination of multiple reprogramming factors, such as pCXLE-hUL,pCXLE-hSK, pCXLE-hOCT3/4-shp53-F, and pEP4 EO2S T2K.

In other embodiments, the reprogramming factors are delivered bytechniques known in the art, such as nuclefection, transfection,transduction, electrofusion, electroporation, microinjection, cellfusion, among others. In other embodiments, the reprogramming factorsare provided as RNA, linear DNA, peptides or proteins, or a cellularextract of a pluripotent stem cell.

In different embodiments, the reprogramming media includes at least onesmall chemical induction molecule. In different embodiments, the atleast one small chemical induction molecule includes PD0325901,CHIR99021, HA-100, A-83-01, valproic acid (VPA), SB431542, Y-27632 orthiazovivin (“Tzv”). In different embodiments, culturing the LCs in areprogramming media is for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or16 days. In different embodiments, culturing the LCs in a reprogrammingmedia is for at least 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days. Indifferent embodiments, culturing the LCs in an induction media is for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

In certain embodiments, the LCL-iPSCs are derived from lymphoblastoidB-cells previously isolated from a subject, by for, example, drawing ablood sample from the subject. In other embodiments, the LCs areisolated from a subject possessing a disease mutation. For example,subjects possessing any number of mutations, such as autosomal dominant,recessive, sex-linked, can serve as a source of LCs to generateLCL-iPSCs possessing said mutation. In other embodiments, the diseasemutation is associated with a neurodegenerative disease, disorder and/orcondition. In other embodiments, the disease mutation is associated withan inflammatory bowel disease, disorder, and/or condition.

This includes, for example, patients suffering from inflammatory boweldiseases and/or conditions, such as ulcerative colitis and Crohn'sdisease. Thus, in one embodiment, iPSCs are reprogrammed from apatient's cells, i.e. are derived from a patient, e.g. with IBD,transformed to organoids, then seeded as single cell suspensions on amicrofluidic chip in order to generate IBD on a chip, see outline ofprogression form lymphoidblastoid B-cell lines to iPSCs (LCL-iPSCs) thenintestinal organoids to IBD on a chip.

However, it is not intended that intestinal cells used on microfluidicchips be limited to cellular sources from IBD patients, in fact, sourcesof white blood cells or other cells for use in providing iPSCs for usein providing intesntial organoids include but are not limited to,patients/subjects having ulcerative colitis and Crohn's disease.

In various embodiments, the LCL-iPSCs possess features of pluripotentstem cells. Some exemplary features of pluripotent stem cells includingdifferentiation into cells of all three germ layers (ectoderm, endoderm,mesoderm), either in vitro or in vivo when injected into animmunodeficient animal, expression of pluripotency markers such asOct-4, Sox-2, nanog, TRA-1-60, TRA-1-81, SSEA4, high levels of alkalinephosphatase (“AP”) expression, indefinite propagation in culture, amongother features recognized and appreciated by one of ordinary skill.

Other embodiments include a pharmaceutical composition including aquantity of lymphoid-cell derived induced pluripotent stem cellsgenerated by the above described methods, and a pharmaceuticallyacceptable carrier.

EXPERIMENTAL

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Allen et al., Remington: The Science and Practice of Pharmacy22^(nd) ed., Pharmaceutical Press (Sep. 15, 2012); Homyak et al.,Introduction to Nanoscience and Nanotechnology, CRC Press (2008);Singleton and Sainsbury, Dictionary of Microbiology and MolecularBiology 3^(rd) ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006);Smith, March's Advanced Organic Chemistry Reactions, Mechanisms andStructure 7^(th) ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton,Dictionary of DNA and Genome Technology 3^(rd) ed., Wiley-Blackwell(Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A LaboratoryManual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor,N.Y. 2012), provide one skilled in the art with a general guide to manyof the terms used in the present application. For references on how toprepare antibodies, see Greenfield, Antibodies A Laboratory Manual2^(nd) ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013);Köhler and Milstein, Derivation of specific antibody-producing tissueculture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July,6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No.5,585,089 (1996 December); and Riechmann et al., Reshaping humanantibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

EXAMPLE 1 Study Design

Described herein is the use of human induced pluripotent stem cells(hiPSCs) to elucidate the adverse effects and mechanisms of chroniclow-dose EDC exposures on developing gut and hypothalamicneuropeptidergic neurons, and serves as a platform for mimicking the inutero exposure to EDCs. Such a screening platform can not onlyfaithfully mimic a human model of development but also can provideinvaluable insights on the developmental cues that could be disrupted bythe compounds screened for.

EXAMPLE 2 Foregut Epithelium Differentiation (iFGE)

For differentiation, iPSCs were accutase-treated and plated into a6-well Matrigel-coated dish at a density of 1 million per well in E8medium with ROCK-inhibitor Y27632 (10 μM; Stemgent). On the next day,iPSCs were differentiated into definitive endoderm by exposing them toActivin A (100 ng/ml; R&D) and Wnt3A (25 ng/ml only on the first day;Peprotech) in RPMI 1640 (Gibco) for 3 days. During these 3 days, thecells were exposed to increasing concentrations of 0%, 0.2% and 2%defined FBS (dFBS, Hyclone). After definitive endoderm induction, thecells were directed to form foregut spheroids by culturing them for thenext 3 days in Advanced DMEM/F12 medium (Gibco) containing 2% dFBS, 2 μMCHIR99021 (2 μM; Cayman), FGF4 (500 ng/ml; Peprotech), LDN (2 μM;Cayman) and retinoic Acid (2 μM; Cayman). This resulted in semi floatingspheroids, which were then selectively picked and transferred on toMatrigel-coated experimental plates for further maturation andexperimentation. For maturing the picked foregut spheroids, they werecultured in a medium containing Advanced DMEM/F12 with N2 (Invitrogen),B27 (Invitrogen), Glutamax, Penicillin/streptomycin/Antimycotic and EGF(100 ng/ml; Peprotech). Media was replaced every 2-3 days as necessaryand the spheroids are allowed to develop into an epithelial monolayeruntil Day 20.

EXAMPLE 3 Hypothalamic Neuron Differentiation (iHTN)

For differentiation into iHTNs, iPSCs were accutase-treated and platedas single cells in 6-well Matrigel-coated plates at a density of approx.1 million cells/well in E8 medium with ROCK-inhibitor Y27632 (10 μM;Stemgent). The next day iHTN differentiation was initiated byneuroectoderm differentiation by dual SMAD inhibition using LDN193189 (1μM, Cayman) and SB431542 (10 μM, Cayman) and this treatment is carriedon for 48 hours. This was followed by Sonic hedgehog activation bySmoothened agonist SAG (1 μM, Tocris) and purmorphamine (PMN, 1 μM,Tocris) and Wnt signaling inhibition using IWR-endo (10 μM, Cayman) fromDay 3 to day 8 to direct the cells towards ventral diancephalon withregular media change every 2 days. Day 9 to Day 13 the cells are slowlymade to exit cell cycle using DAPT (10 μM, Cayman) in the presence ofventralizing agent retinoic acid (0.1 μM, Cayman). On Day 14, the cellswere accutased and replated onto Laminin-coated plates in the presenceof maturation medium containing brain-derived neurotrophic factor BDNF(10 ng/ml, Miltenyi) and maintained until Day 40.

EXAMPLE 4 EDC Treatments

The Inventors employed 3 different EDCs, Perfluorooctanoic acid (PFOA)(2.5 μM, Sigma-Aldrich), Tributyltin (TBT) (10 nM, Sigma-Aldrich) andButylated hydroxytoluene (BHT) (10 nM, Cayman) individually and incombination. The Inventors hence had 6 treatment groups namely Vehiclecontrol (Vh), PFOA, TBT, BHT and combination treatment. iFGE treatmentof EDCs was carried out by performing the differentiation as mentionedabove and adding EDC treatments during the final 12 days ofdifferentiation i.e. Day 8 to Day 20. Similarly, iHTNs weredifferentiated as per the protocol detailed above and the final 12 daysof differentiation i.e. Day 28 to Day 40 EDC treatments were performed.For the rescue experiments using NFicBi (SN50), the cells were firstexposed to NFκBi 24 hours prior to EDC treatment. Subsequently, thecells were treated with the combination treatment along with NFκBi. Itshould be noted that that NFκBi treatment was only combined withcombination EDC treated conditions.

EXAMPLE 5 Immunofluorescence

Cells that were subject to immunofluorescence were first fixed using 4%paraformaldehyde (PFA) for 20 minutes and subsequently washed with PBS.After blocking the cells with 5% donkey serum (Millipore) with 0.2%triton X-100 (Bio-rad) in PBS for a minimum of 2 hours, the cells werethen treated with an appropriate concentration of relevant primaryantibody combinations (1:250) overnight at 4° C. After thorough washingusing PBS with 0.1% Tween-20, the cells are then treated withappropriate species-specific Alexa Fluor-conjugated secondary antibodycombinations for 45 minutes (1:500). Hoechst stains were used to markthe nuclei and the cells were then visualized using appropriatefluorescent filters using ImageXpress Micro XLS (Molecular devices).

EXAMPLE 6 Immunoblots

Cell pellets were collected and lysed (mammalian PER, Thermoscientific+1× protease inhibitor cocktail, Thermo Scientific) andsamples were prepared after protein quantification. The Inventors loadedabout 15 μg protein per lane of a polyacrylamide gel (NuPAGE™ Novex™4-12% Bis-Tris Protein Gels). Once the gels were resolved, they weretransferred onto nitrocellulose membrane and subsequently blocked in 5%milk solution for a minimum of 2 hours. This was followed by a one-stepi-Bind process which treated the membrane with primary antibody, washingand secondary antibody steps (Life technologies). The Inventors employedLiCor® IRDye secondary antibodies (680 and 800 wavelength infrared dyes)and detection of bands was carried out in a LiCor ODyssey CLx imager(Li-Cor).

EXAMPLE 7 Quantitative PCR

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and RNA (2 μg)was first DNase treated and reverse transcribed to cDNA with oligo(dT)using the Promega Reverse Transcriptase System (Promega). Reactions wereperformed in three replicates using SYBR Green master mix (AppliedBiosystems) using primer sequences specific to each gene. Each PCR cycleconsisted of 95° C. for 10 minutes, 95° C. 30 seconds →58° C. for 60seconds, for 50 cycles, and 72° C. for 5 minutes. Genes of interest werenormalized to either RPL13A or 16srRNA for mitochondrial genes.

EXAMPLE 8 MTT Assay

Cell viability was assessed by MTT assay. Cells were plated in 96-wellplates at a density of 10,000 cells in 100 μL medium per well. On theday of assay, fresh media was added (100 μL) and 10 μL MTT solution wasadded to the culture medium (12 mM stock MTT solution) and incubated at37° C. for 4 hours. The reaction was stopped by the addition of 50 μLDMSO to each well. A no cell negative control was included to subtractbackground. The absorbance value was read at 540 nm using an automaticmulti-well spectrophotometer (Perkin Elmer).

EXAMPLE 9 Metabolic Phenotyping and Seahorse Respirometry Assay

The Seahorse XF^(e)24 Extracellular Flux Analyzer (Seahorse Biosciences)was used to perform mitochondrial stress tests and obtain real-timemeasurements of oxygen consumption rate (OCR) in cells. iFGEs and iHTNStreated with or without EDCs were seeded in a 24-well Seahorse cultureplate at a density of 10,000-15,000 cells/well. For analysis of OCR,cells were reconstituted in Seahorse base medium and were allowed tosettle for 1 hour at 37° C. in non-CO₂ incubator before measurements.Chemical reagents (Sigma) were used at final concentrations as follows:1 μM Oligomycin—an ATP synthase inhibitor, 1 μM (FCCP) carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone—an uncoupling agent, and a mixtureof 0.5 μM antimycin A—a cytochrome C reductase inhibitor and 0.5 μMrotenone—a complex I inhibitor. Results were normalized to proteinconcentration determined by BCA assay (Thermo Scientific).

EXAMPLE 10 Statistical Analysis

All data are represented as mean±SD or SEM. p<0.05 was consideredsignificant. All statistical analyses were performed on Graphpad Prismusing student's paired t-test or one-way Analysis of variance (ANOVA)and Newman-Keuls post-test for multiple comparisons.

EXAMPLE 11 Primary and Secondary Antibodies

Immunocytochemistry staining: Primary: α-MSH, rabbit, PhoenixPharmaceuticals, H-43-01, 1:250; β-catenin, rabbit, Santa Cruz, sc7199,1:500; CART, goat, Santa Cruz, sc18068, 1:250; CPE, goat, R&D Systems,AF3587, 1:250; E-cadherin, goat, R&D Systems, AF648, 1:250; GABA,rabbit, Sigma-Aldrich, A2025, 1:250; Gastrin, rabbit, Dako, A056801-2,1:250; Ghrelin, goat, Santa Cruz, sc10368, 1:250; NF-κB (PhosphoSer-311), mouse, Santa Cruz, sc166748, 1:250; NP-II, goat, Santa Cruz,sc27093, 1:250; NPY, rabbit, MerckMillipore, AB9608, 1:250; OTP, rabbit,Genetex, GTX119601, 1:250; Peptide YY, rabbit, Abcam, ab22663, 1:250;Serotonin, rabbit, Immunostar, 20080, 1:250; Somatostatin, rabbit, SantaCruz, sc13099, 1:250; Sox17, mouse, Novus, 47996, 1:250; Sox2, rabbit,Stemgent, 09-0024, 1:500; Synaptophysin, mouse, Santa Cruz, sc17750,1:250; TH, mouse, Immunostar, 22941, 1:250. Secondary (1:200):AlexaFluor 488 donkey anti-rabbit, AlexaFluor 555 donkey anti-mouse,AlexaFluor 594 donkey anti-mouse, AlexaFluor 568 donkey anti-goat,AlexaFluor 647 donkey anti-goat.

Immunoblotting: COX IV, rabbit, Cell Signaling, 4850, 1:2000; NF-κB p65(Phospho Ser-311), mouse, Santa Cruz, sc166748, 1:1000; NF-κB p65(RelA), rabbit, Cell Signaling, 8242, 1:1000; NF-κB1 (p105/p50), CellSignaling, 12540, 1:1000; NF-κB2 (p100/p52), Cell Signaling, 4882,1:1000; Phospho p53 (Ser15), rabbit, Cell Signaling, 9284T, 1:500; p53,rabbit, Cell Signaling, 9282T, 1:500; IRE160 , rabbit, Cell Signaling,3294, 1:500; Ero1, rabbit, Cell Signaling, 3264, 1:500; BiP, rabbit,Cell Signaling, 3177, 1:500.

Secondary (1:2000): IRDye 800CW, donkey anti-rabbit, Li-Cor, 926-32213;IRDye 680LT, donkey anti-mouse, Li-Cor, 926-68022.

EXAMPLE 12 Primer Sequences

AGRP Forward (SEQ ID NO: 1) 5′-GGATCTGTTGCAGGAGGCTCAG-3′, Reverse(SEQ ID NO: 2) 5′-TGAAGAAGCGGCAGTAGCACGT-3′; CDX2 Forward (SEQ ID NO: 3)5′-CTGGAGCTGGAGAAGGAGTTTC-3′, Reverse (SEQ ID NO: 4)5′-ATTTTAACCTGCCTCTCAGAGAGC-3′; GKN1 Forward (SEQ ID NO: 5)5′-CTTTCTAGCTCCTGCCCTAGC-3′, Reverse (SEQ ID NO: 6)5′-GTTGCAGCAAAGCCATTTCC-3′; MC4R Forward (SEQ ID NO: 7)5′-CTTATGATGATCCCAACCCG -3′, Reverse (SEQ ID NO: 8)5′-GTAGCTCCTTGCTTGCATCC-3′; NKX2-1 Forward (SEQ ID NO: 9)5′-AACCAAGCGCATCCAATCTCAAGG-3′, Reverse (SEQ ID NO: 10)5′-TGTGCCCAGAGTGAAGTTTGGTCT-3′; NPY Forward (SEQ ID NO: 11)5′-GGTCTTCAAGCCGAGTTCTG-3′, Reverse (SEQ ID NO: 12)5′-AACCTCATCACCAGGCAGAG-3′; OPRM1 Forward (SEQ ID NO: 13)5′-TGGTGGCAGTCTTCATCTTG-3′, Reverse (SEQ ID NO: 14)5′-GATCATGGCCCTCTACTCCA -3′; PDX1 Forward (SEQ ID NO: 15)5′-CGTCCGCTTGTTCTCCTC-3′, Reverse (SEQ ID NO: 16)5′-CCTTTCCCATGGATGAAGTC-3′; PGA5 Forward (SEQ ID NO: 17)5′-CCATCTTGCCTTCTCCCTCG-3′, Reverse (SEQ ID NO: 18)5′-TCTGATGAGGGGGACCTTGT-3′; SOX2 Forward (SEQ ID NO: 19)5′-TTC ACA TGT CCC AGC ACT ACC AGA-3′, Reverse (SEQ ID NO: 20)5′-TCA CAT GTG TGA GAG GGG CAG TGT GC-3′; TAS1R3 (SEQ ID NO: 21)Forward 5′-ACGTCTGACAACCAGAAGCC-3′, Reverse (SEQ ID NO: 22)5′-CAGTCCACACAGTCGTAGCA-3′; TFF1 Forward (SEQ ID NO: 23)5′- TGGAGGGACGTCGATGGTAT-3′, Reverse (SEQ ID NO: 24)5′-TGGAGGGACGTCGATGGTAT-3′; TFF2 Forward (SEQ ID NO: 25)5′-CTGAGCCCCCATAACAGGAC-3′, Reverse (SEQ ID NO: 26)5′-ACGCACTGATCCGACTCTTG-3′; Large mito Forward (SEQ ID NO: 27)5′-TCTAAGCCTCCTTATTCGAGCCGA-3′, Reverse (SEQ ID NO: 28)5′-TTTCATCATGCGGAGATGTTGGATGG-3′; Small mito Forward (SEQ ID NO: 29)5′- CCC CAC AAA CCC CAT TAC TAA ACC CA-3′, Reverse (SEQ ID NO: 30)5′-TTTCATCATGCGGAGATGTTGGATGG-3′; β-globin Forward (SEQ ID NO: 31)5′-CGA GTA AGA GAC CAT TGT GGC AG-3′, Reverse (SEQ ID NO: 32)5′-GCA CTG GCT TAG GAG TTG GAC T-3′. HPRT Forward (SEQ ID NO: 33)5′-TGG GAT TAC ACG TGT GAA CCA ACC-3′, Reverse (SEQ ID NO: 34)5′-GCT CTA CCC TCT CCT CTA CCG TCC-3′.

EXAMPLE 13 Peripheral Blood Mononuclear Cells are EpisomallyReprogrammed to Pluripotency

Non-integrating reprogramming of peripheral blood mononuclear cells(PBMCs) to iPSCs was performed using the episomal (OriP/EBNA1)plasmid-based method similar to published protocols in the Inventors'lab (FIG. 8A). This included nuclear transfection of seven episomallyexpressed reprogramming factors OCT3/4, SOX2, KLF4, LIN28,non-transforming L-MYC, SV40 large T antigen (SV40LT) and shRNA againstp53. This protocol resulted in successful generation of blood-derivednon-integrating iPSC clones that could be mechanically isolated andexpanded after 27-32 days (FIG. 8A). Representative images fromindependent donor-derived iPSC lines used in this study (80iCTR Tn2 and201iCTR NTn4) exhibited typical features of pluripotent stem cells suchas tight colonies with high nucleus to cytoplasm ratio as shown bybright field images on FIG. 8B. They also showed a robust alkalinephosphatase activity, exhibited strong expression of nuclear (OCT3/4,NANOG, SOX2) and surface (SSEA-4, TRA-1-81, TRA-1-60) pluripotencyproteins (FIG. 8B). The PBMC-iPSCs generated also passed the PluriTestassay with high pluripotency and low novelty scores (FIG. 8C) andmaintained normal cytogenetic status as shown by G-band karyotypespreads (FIG. 8D).

EXAMPLE 14 Human iPSCs Differentiate into Endocrinally Active ForegutEpithelium (iFGE) by Modulation of WNT, FGF, BMP and Retinoic AcidSignaling

Based on the 3-D gastric organoid differentiation previously publishedby the Wells group where they employed a three dimensional matrigelbubble to mature the stomach organoids, the Inventors employed amodification of their protocol to generate two dimensional monolayers ofgastric epithelium with endocrine abilities. The specification of iPSCinto antral foregut epithelium, containing endocrine cell types wassuccessfully achieved in a stepwise method by; (1) Activin A andWnt3A-mediated definitive endoderm specification, (2) simultaneousactivation of WNT (CHIR), FGF (FGF4) and Retinoic Acid (RA) signalingwhile repressing BMP signaling, and (3) final generation of endocrinecell containing foregut epithelium with high concentrations of epidermalgrowth factor (EGF) (FIG. 1A). After definitive endoderm induction, at 6days post-iPSC, gut-tube like organoid structures emerge from theendoderm monolayer. Upon re-plating the gut-tube organoids, an adherentepithelial-shaped cell layer consistently emerges between 7 and 20 dayspost-iPSCs (FIG. 1B). The characterization of iPSC-derived foregutepithelium (iFGE) at day 20 was confirmed by monitoring expression ofrelevant stomach/foregut-specific genes. Significant expression of SOX2(foregut progenitor), PDX1 (antral foregut), GKN1 (gastrokine 1; gastricmucosa), PGA5 (digestive enzyme), TAS1R3 (taste receptor in the foregut)and TFF2 (trefoil factor 2; stable secretory protein of gastric mucosa)genes expressed in the foregut were observed in day 20 adherent iFGEs(FIG. 1C). It is important to note that the iFGEs did not exhibitexpression of hindgut-specific CDX2 (FIG. 1C). Upon evaluating forepithelial cell surface-specific proteins, CDH1 (E-cadherin) and CTNNB(β-catenin), were regularly observed at the surface in polygonalcobblestone shaped cells, as sheets of iFGEs formed (FIG. 1D). Endodermand foregut progenitor-specific transcriptional factors, Sox17 and Sox2,respectively, confirm the foregut identity of the iFGEs (FIG. 1D).Importantly, neuroendocrine markers known to be present in endocrinallyactive foregut such as synaptophysin (SYP), somatostatin and serotoninwere expressed by the iFGE at day 20 (FIG. 1E). Notably, the iFGEs werealso immunopositive for stomach-specific hormone-expressingenteroendocrine cells like gastrin (G cells), ghrelin (parietal cells)and a few peptide YY (mucosal) cells (FIG. 1F).

EXAMPLE 15 Functional Neuropeptidergic Hypothalamic Neurons (iHTNs) canbe Derived from hiPSC-Neuroepithelium by Activating SHH and InhibitingWNT Signaling

The iHTNs were generated after directed patterning and neuroepitheliumspecification with dual SMAD inhibition (SMADi) small molecule treatmentof iPSCs. Subsequently, early WNT inhibition and SHH activationspecified forebrain cell types of ventral diencephalon identity wherethe hypothalamus and the arcuate nucleus resides (FIG. 2A).Synchronizing the forebrain progenitors and terminal maturation of thedifferentiating neurons by day 40 yields increased expression ofhypothalamic and neuropeptidergic genes such as AgRP (Agouti-relatedPeptide; an orexigenic neuropeptide), MC4R (Melanocortin 4 receptor;regulation of feeding and metabolism), Nkx2.1 (ventral diencephalonmarker), NPY (Neuropeptide Y; orexigenic neuropeptide co-expressed withAgRP), and PCSK2 (Proprotein Convertase Subtilisin/Kexin Type 2;neuroendocrine gene) (FIG. 2B). The secretion of critical hypothalamicneuropeptides NPY and α-melanocyte-stimulating hormone (α-MSH) wasconfirmed using ELISA and results revealed significantly higher levelsof both neuropeptides in day 40 iHTNs (FIG. 2C and FIG. 2D).Immunofluorescence staining showed neurons expressing severalneuroendocrine and hypothalamic arcuate nucleus-specific proteins likeOTP (homeobox protein orthopedia; FIG. 2E), a-MSH (FIG. 2F), NPY (FIG.2G), SST (somatostatin; FIG. 2H), GABA (FIG. 2I), CPE (carboxypeptidaseE; FIG. 2J), CART (Cocaine- and amphetamine-regulated transcript; FIG.2K), NP-II (neurophysin II/arginine vasopressin; FIG. 2L), 5-HT(serotonin; FIG. 2M) and TH (tyrosine hydroxylase; FIG. 2N).Electrophysiological measurements using multi-electrode array (MEA)platform shows regular trains of spontaneous action potentials andrepetitive firing in day 40 neurons when compared to no activity at day0 stage, thus confirming bona fide neuronal identity and electricalmaturity of iHTNs (FIG. 2O).

EXAMPLE 16 Chronic Low-Dose EDC Treatment Perturbs NF-κB signaling iniFGEs and iHTNs Without Affecting Cell Viability

After successful differentiation of iPSC-endocrine cell cultures, theInventors decided to perturb these tissues with EDCs at low-dose over atwelve-day treatment paradigm. The optimal concentrations for EDCtreatments were determined as log or semi-log concentration below thedose at which even a 10% loss in cell viability was observed in thedifferentiated iPSC-endocrine cultures (FIG. 9A-9F). Additionally,literature search was utilized to know human tolerable daily intake(TDI) and the effect of a range of each of the compounds on cellviability was performed and in accordance individual treatments withperfluoro-octanoic acid (PFOA; 2.5 μM), tributyltin (TBT; 10 nM) andbutyl hydroxytoluene (BHT; 10 nM) were given, along with combinationtreatment paradigm that is similar to concomitant environmental exposureto multiple EDCs. Upon treatment with EDCs in developing iPSC-derivedendocrine tissues, a significant increase in phosphorylated NF-κB p65immunopositive cell numbers was observed in iFGE cells from 1.35 to1.5-fold (FIG. 3C) and 1.2 to 1.3-fold in iHTNs (FIG. 3D) (p<0.001).Immunoblotting of these cultures confirmed that NF-κB p65phosphorylation levels were shown to be significantly elevated inEDC-treated iFGEs (FIG. 3E) (p<0.001) and iHTNs (FIG. 3F) (p<0.01). Toconfirm that the addition of EDCs and the resulting increases in NF-κBphospho-p65 is not a consequence of EDC-induced loss in cell viabilityor general cytotoxicty, an MTT cell viability assay on the EDC-treatedand vehicle-treated iFGEs and iHTNs was performed. It was found thatthese treatments EDCs did not affect significantly affect cell viabilityin both iPSC-derived tissue types (FIGS. 3G and 3H).

Phosphorylation of NFKB p65 is part of its activation process andwell-known to be associated with deleterious pro-inflammatory activationpathways in blood cells. Phosphorylation is required for dimerizationwith p50 and translocation to the nucleus. Since p65 (RelA) activationwas observed with EDC treatment, activation of the canonical NF-κBpathway was assessed by determining the ratio of the active p50 form tothe inactive p105 (NFK31) subunit. The dimerization of p50 with thephosphorylated p65 subunit and subsequent proteasomal degradation ofIκBα leads to the typical nuclear translocation of p65-p50 dimersresults in the transcriptional regulation of κB-dependent genes (FIG.4A). Upon individual and combination EDC treatments, p50 levels werehigher in relation to its precursor p105 (FIG. 4B), which showsactivation of the canonical pathway in EDC treated iPSC-endocrinecultures. Interestingly, both iFGEs and iHTNs showed EDC-mediatedincrease in p50/p105, where iFGEs displayed a 2 to 3-fold increase(p<0.001) (FIG. 4B), while iHTNs showed 1.5-2 fold increase (p<0.001)(FIG. 4E). In a similar approach to determine the involvement/activationof the non-canonical NF-κB pathway, the Inventors measured the ratio ofprotein expression of p100 to p52. Briefly, the non-canonical NFκBpathway involves the dimerization of ReIB and p52 and hence a measure ofthe amount of p52 provides a measure of the possible activation of thispathway (FIG. 4D). Similarly, the Inventors also observed significantincreases in the ratio of p52/p100 with the treatment of EDCs in bothiFGE (1.4 to 2-fold; p<0.001) and iHTN (1.5 to 2.5-fold; p<0.001) (FIG.4F). Thus, for the first time the Inventors demonstrated that EDCsmediate their action on developing human endocrine cells bysignificantly perturbing the NF-κB pathway.

EXAMPLE 17 EDCs Impinge on Metabolic Activity by DisruptingMitochondrial Respiration

Because one of the Inventors' aims was to determine whether chronic EDCperturbation effects metabolic activity and respiration in humanendocrine tissues, the Inventors also inquired how NF-κB phosphorylationmay also contribute to this phenomenon. Interestingly, there is someevidence in cancer biology where NF-κB signaling influencesmitochondrial function, both by directly and indirectly regulatingtranscription of relevant nuclear- and mitochondrially-encodedrespiratory genes. First, the Inventors determined the effects of EDCson mitochondrial respiratory function by performing a mitochondrialstress test with an XF^(e)24 Seahorse Extracellular Flux Analyzer. TheInventors determined that in iFGEs the addition of BHT (p<0.05) and acombination treatment of PFOA, TBT and BHT (p<0.01) brought about adecrease in maximal respiration and spare respiratory capacity by 40-50%(FIG. 5A). Exhibiting a similar effect in the iHTNs, treatment with TBT,BHT and the combination treatment again showed a 40-50% decrease inmaximal respiration and spare respiratory capacity (FIG. 5B). The effectof treatments on mitochondrial mass was ruled out since the COX IV(inner mitochondrial membrane enzyme) levels between all treatments didnot vary (FIG. 5C and FIG. 5D).

In an attempt to deduce possible transcriptional regulation of thisimpairment in mitochondrial function, the Inventors examined the geneexpression levels of critical nuclear-encoded mitochondrial respiratorygenes such as SCO2 (Cytochrome C oxidase 2), POLRMT (Mitochondrial RNApolymerase), TFAM (transcription factor A, mitochondrial) andmitochondrially-encoded CYTB5 (Cytochrome B5). Both iFGEs and iHTNs weresignificantly impacted by EDC treatments, as critical respiratory geneslike SCO2, POLRMT, TFAM, and CYTB5 were down regulated as a result ofindividual EDC treatment, with combination treatment engendering mostsignificant decrease in mRNA levels (FIGS. 5C-5F).

EXAMPLE 18 NF-κB Inhibition Rescues Cells from Pathway Activation andMitochondrial Impairment

Considering that the adverse NF-κB pathway perturbation andmitochondrial dysfunction effects due to EDC exposure was pronounced inthe developing iPSC-endocrine cultures, the Inventors explored whetherthese phenotypes can perhaps be rescued by simply blocking NF-κB pathwayactivation. Therefore, the Inventors employed a NF-κB inhibitor (NFκBi)SN50, a cell permeable inhibitory peptide, to determine whether this canrescue the previous phenotypes in iPSC-endocrine cultures treated withthe deleterious combination EDC treatment. SN50 peptide that is known toinhibit nuclear induction of the NF-κB regulatory genes. Uponco-treatment with EDCs and NF-κBi in the iFGEs, the Inventors found anoverall decrease in phospho-p65, canonical (p50/p105) and non-canonical(p52/p100) pathway almost returning to the levels of the vehicle control(p<0.001). NF-κBi did not appear to confer a specific inhibitory effecton p50 alone, but rather a more generic inhibitory effect on activatedp65, p50 and p52 levels (FIG. 6A) compared to combination treatmentalone. This rescue effect of SN50 NFκBi treatment was also confirmedwhen immunopositive pNF-κB cells decreased close to vehicle controllevels (FIG. 6B). Particularly, NF-κBi treatment also significantlyimproved the mitochondrial spare respiratory capacity of the combinationEDC treated cells (FIG. 6C). The finding that was of the most interestis that the transcriptional regulation of proteins involved inmitochondrial function such as SCO2, POLRMT, TFAM, and CYTB5 were allrestored upon NFκBi treatment compared to EDC combination treatment(FIG. 6D). These results were reproduced in the iHTNs where NF-κBitreatment significantly reversed combination EDC treatment-mediatedeffects (FIG. 7D). This novel finding linking NF-κB pathway perturbationto severe mitochondrial dysfunction has not been demonstrated in anysystem, especially in the context of endocrine disruption.

EXAMPLE 19 Discussion

According to the “environmental obesogen” hypothesis, a subset ofpervasive environmental pollutants, known as endocrine disruptingchemicals (EDCs), target hormonal signaling pathways, disrupting normaltissue development and interfere with the body's homeostatic controls.Repeated exposures of ubiquitous “obesogenic” EDCs like organotins,perfluorochemicals, and food additives mainly through human food duringcritical windows of stem cell development in utero or early-life couldadversely alter some genetically pre-disposed individuals' normalmetabolic control permanently, setting them up for obesity later inlife. Noteworthy is the fact that these EDCs continue to be present inthe Inventors' daily environments and continue to pose health hazards. Arecent article revealed the presence of PFOA in drinking water sourcedfrom the Tennessee river despite efforts on phasing out the use of PFOAas per EPA's request (Environmental protection Agency). Similarlyefforts to remove BHT as an additive in cereals have been put forwardand major brands have successfully removed BHT as an additive from theircereals. Given that a daily exposure to these EDCs keep exposing us toendocrine disruption and related effects, the impact of these EDCs needto be studied in better details.

Barring a few specific instances of obesity arising from traceablegenetic causes, a slew of biological and behavioral factors affectenergy balance. The genetic basis has been extensively investigated andgenome-wide association studies (GWAS) have identified many obesityassociated loci. However, only a small percentage of these can either beexplained or validated in animal models. Assuming that the Inventors'human gene pool has not changed as expeditiously as the upsurge inchildhood obesity, the modern chemical environment interacting with anindividual's genetic background, is the likely driving mechanismpromoting this risk for and modifying the severity of obesity. Betterbiomarkers and mechanisms predicting the manifestations of pervasiveEDCs interfering with endocrine functions in developing human tissuesare lacking at least partially due to paucity of appropriate humancellular models to probe gene-environment interactions. The Inventorsdecided to address these gaps using pluripotent stem cells where theInventors posited that chronic exposure of low-dose EDCs to humaniPSC-endocrine cells is detrimental to early endocrine tissuedevelopment, via hyperactive NF-κB signaling and mitochondrialdysfunction, possibly contributing to metabolic diseases like obesityand type 2 diabetes.

Metabolic changes during developmental programming have been of greatinterest in recent years. With the increasing prevalence of obesity inchild-bearing individuals, the developmental programming of the fetuscan be subject of alterations in organ formation and tissue development,metabolism and predisposition of offspring to metabolic disorders. Inthe Inventors' current work, the Inventors investigate the detrimentaleffects of exposure to putative endocrine disrupting chemicals indeveloping cells i.e. iHTNs and iFGEs. The Inventors' in vitro datareveal that EDC treatment in both iFGEs and iHTNs bring about anincrease in phosphorylated p65. p65/RelA is part of the classicalcanonical NFKB pathway that is known to be stimulated by cytokines suchas tumor necrosis factor-a (TNF-a) or other infectious agents, anddepends on the degradation of IκB via its ubiquitination which leads top65:p50 dimers thereby activating this pathway. In general, the NFκBpathway has been fairly well studied in cancer biology and tumorprogression, but little is known with regard to its role indevelopmental programming and metabolism. However, it is noteworthy thatin the presence of low-dose EDCs, the Inventors observed increasedphosphorylation of p65 in endocrine tissues, iHTNs and iFGEs. Thisadverse perturbation of NFκB suggests greater retention and long termeffects of EDCs in the neural tissue and could implicate effects onneuroendocrine and food-intake circuitries as well as brain development.To confirm long-term direct developmental effects of EDCs on mammalianstomach and brain, further studies are warranted in either animal modelsor human cells.

Similarly, the Inventors found increased processing of p105 to p50 aswell as p100 to p52 in both iFGE and iHTNs with EDC treatments. This wasan interesting finding since EDCs have shown activation of both thecanonical and non-canonical pathways with EDCs. Studies have suggestedthat p65/RelA could be involved in transactivation of both p105 and p100promoters. Hence RelA could be a common activator of both the canonicaland non-canonical pathways of NFKB. NF-κB has been pointed towardsinfluencing mitochondrial function via crosstalk through the abovementioned proteins.

One of the interesting aspects of this study was revealed when theInventors found differences in mitochondrial respiratory capacity iniHTNs and iFGEs in the presence of EDCs. However the Inventors foundvarying degrees of effect of each EDC on mitochondrial respiratorycapacity. In the iFGEs, only BHT and combination treatments broughtabout a significant decrease in the spare respiratory capacity, whereasin the iHTNs, TBT, BHT and combination treatment brought about asignificant impairment in spare respiratory capacity. PFOA treatment inboth cell types did not show any effect. Impairment in mitochondrialspare respiratory capacity would translate into either increased basalrespiration rate, increased proton leak or a decrease in maximalrespiratory capacity of the mitochondria. To test whether the impairmentin mitochondrial capacity translated to differences in transcription ofgenes involved in mitochondrial function, the Inventors measured mRNAlevels of 4 proteins involved in mitochondrial function namely: a) SCO2(subunit of cytochrome c oxidase), b) POLRMT (Mitochondrial RNApolymerase), c) TFAM (Transcription factor A, mitochondrial) all ofwhich are nuclear encoded and d) CytB5 (Cytochrome B 5) which ismitochondrially encoded.

It was interesting to note that all these genes were down regulated uponEDC treatment in both iHTNs and iFGEs. This might explain the impairmentin mitochondrial function in the presence of EDCs. Some have proposed apossible mechanism through which NFκB regulates ATP production viaaffecting both nuclear and mitochondrial gene expression. They proposethat a crosstalk between NFκB RelA and certain transcription factorsregulate expression of nuclear encoded mitochondrial proteins such asTFAM and POLRMT. Furthermore they suggest that RelA could also directlybe translocated to mitochondria and repress mitochondrial geneexpression thereby contributing to downregulation of oxidativephosphorylation. One study reported that RelA knockdown lead toincreased binding of POLRMT to the D-loop of mitochondrial genome,increased Cytochrome B mRNA levels and increased ATP production. Takentogether, these findings support the Inventors' data that increased RelAbrings about decreases in Cytochrome B5 mRNA levels. Additionally, giventhe Inventors' observation of decreased mitochondrial respiration anddecreased POLRMT mRNA levels upon RelA activation, ATP production in theInventors' EDC treated cells may also be presumably attenuated.

In an attempt to elucidate if suppressing NFκB would reverse theseeffects and if the impairment of mitochondrial function is linked to theactivation of NFκB pathway, the Inventors employed an NFκB inhibitor,SN50 (NFκBi). This is a cell permeable peptide which was initially knownto inhibit p50, but was later shown that SN50 was not only specific forp50 but could also affect other NFκB transcription factors In line withthis, the Inventors found that NFκBi treatment significantly decreasedthe EDC treatment mediated increases in phospho p65, p50 as well as p52.Linking NFκB to mitochondrial function, the Inventors also found thatNFκBi treatment restored EDC-mediated decrease in mitochondrial sparerespiratory capacity as well as NFκB target genes.

The involvement of RelA appears to have a critical role in affectingmitochondrial respiration. Certain studies have shown that duringglucose starvation in mouse embryo fibroblasts (MEFs), RelA activatesoxidative phosphorylation and decreases glycolysis. Based on this study,it may be safe to assume that during non-starvation, the glycolyticswitch stays in favor of glycolysis as a source of energy and hence theInventors do not observe an increase in oxidative phosphorylation.However the Inventors notice a decrease in mitochondrial respirationrate and a decrease in genes involved in mitochondrial respiration suchas SCO2, POLRMT, TFAM and CytB5. RelA has been widely argued to becontextual in activating on repressing oxidative phosphorylation andhence the cellular environment and substrate levels may play a majorrole in determining RelA's context in oxidative phosphorylation. In theInventors' study, RelA upon activation by EDCs could possibly actdirectly upon its nuclear targets to repress mitochondrial respirationvia repression of genes involved such as SCO2, POLRMT and TFAM.

Studies have pointed towards recruitment of RelA to mitochondrial genomeand its C-terminal transactivation domain brings about the repression ofPOLRMT binding to mtDNA. CytB5 which is a mitochondrially encoded genehas also been previously shown to be regulated by NFKB. Taken together,the Inventors' data, in part shown in FIGS. 5-7, 9, 12-13, 17-19,suggests that activation of NFκB plays a role in repressingmitochondrial respiration. The functional and developmental implicationsof this effect needs to be probed further. For example, studies directedat observing effects in adipose tissue may further identical roles ofEDC compounds in defining energy homeostasis. Proteomics analysis on theEDC-treated iFGEs and iHTNs and adipocytes would also allow forelucidation of all fragment ions of detectable peptide precursors,thereby aiding the identification of dysregulated proteins.

EXAMPLE 20 Further Studies

Effects of various drugs/compounds/pollutants on fetal development hasbeen an avenue which needs to be addressed urgently in order to avoidbirth defects, developmental defects and to improve overall quality oflife in the coming generations. With global pollution levels constantlyincreasing the inevitable risk of exposure to harmful environmentalpollutants and toxicants is also increasing. There is a need for aconsistent drug screening platform which would provide a clearindication on the effect of these toxicants within the system.

Specially the impact of these compounds on developing fetal tissuescould be even more detrimental as they do not have a fully developedxenobiotic metabolism or immune system to combat the exposed xenobiotic.But obtaining such cells during human fetal development for studyingpotential harmful exposures to various drugs and compounds is highlyimplausible. This invention, however, fills that void by employinghiPSCs to perform directed differentiation of tissues of interest (inthis case endocrine tissues) and study the effect of the toxicants onthe early development of these tissues. This invention as a drugscreening platform can be used to assess the effect of not onlyenvironmental pollutants (EDCs) but also a plethora of prescribed drugs,abused drugs as well as several other compounds whose effects ondeveloping tissues is unknown.

Several drug screening and toxicity testing have been in place in thepast. These drug screening methods include either the use of rat ormouse models or small animal models for toxicity screening in vivo orthe use human cancer lines in vitro to test drug effects. The use of rator mouse models of drug screening has several caveats such asdifferences the animal system has from human system, the animal's ownadaptation to a specific response which could mask the drug's effectwhich may not be present in a human system. The same applies to smallanimal models of drug testing and screening including the use ofnematodes—Caenorhabditis elegans, fruit fly—Drosophila melanogaster orfish Danio rerio. These methods can only be employed as a means toidentify potential targets and pathways to test for in more relevantsystems and come to a conclusion based on collective data.

Similarly cancer line models for drug screening have been an importantplatform but can be considered more relevant for cancer research and thestudy of drug response for treatment of cancer since these lines retaingenetic and epigenetic features of the tumor itself. There is hence aneed for a faithful model that could represent the human system in vitroas well as provide the flexibility to screen for developmental effectsof the compounds and our invention provides a potential platform to doso.

Given several platforms for drug screening currently employed there isstill a need for a drug screening method that could faithfully mimic thehuman system especially during developmental stages has been lacking.Current models for screening use mouse models or tumor/immortalized celllines for screening endocrine dysfuction of many chemicals.

The described technology by the use of hiPS cells which revert normaladult human cells back to a pluripotent stage, and by conferring theability to be directed to almost any tissue/cell type of interest,provides a novel platform to screen for effects of variouscompounds/drugs/toxicants during critical stages of human (fetal orinfant) development. Importantly, hiPSCs also provide and unlimitedsupply of normal progenitor cells from which many relevant and differentendocrine-like tissues can be created from an individual. These cellscan then be used for predictive toxicology and chemical safetyscreening.

Such a screening platform can not only faithfully mimic a human model ofdevelopment but also can provide invaluable insights on thedevelopmental cues that could be disrupted by the compounds screenedfor. The examples below provide designs and exemplary materials, methodsand data for providing models for drug screening and response of cellsto test agents.

EXAMPLE 21 Additional Studies

This example describes exemplary results related to EDC-mediateddysregulation.

Additional experiments related to EDC effects, as described in part inExample 4.

As shown in FIG. 15A-B, (FIG. 15A) iFGE and (FIG. 15B) iHTNs,representative immunoblots showing levels of bona fide ER stress pathwayproteins, IRE1, BiP and Ero1 and Cox IV. Quantified histograms usingImageJ-based densitometry of bands for each of the respective proteinimmunoblots normalized to Cox IV as loading control are shown below andrepresented as fold-change compared to vehicle-treated control. RE1protein increases, while BiP and Ero1 levels decrease in response to EDCexposure, *p<0.05, ** p<0.01, *** p<0.001. All statistical analysis wasperformed using one-way ANOVA. Data shown are representative of averageresults from the two iPSC lines differentiated n=3 times in independentexperiments.

As shown in part in FIG. 4A-F, EDC treatment causes disturbances inNF-κB p65 Canonical and Non-canonical Pathways.

As shown in FIG. 16A-C, Chronic Low-Dose EDC Treatment Perturbs NF-κBsignaling. (FIG. 16A) Top panel: Representative immunocytochemistry(ICC) showing increases in phosphorylated p65 (red) in iFGEs co-stainedwith ghrelin (green); Bottom panel: Representative ICC showing increasesin phosphorylated p65 (red) in iHTNs co-stained with synaptophysin(green). (*** p<0.001). Immunopositive cells were scored and quantifiedinhistograms for both iFGEs and iHTNs, which is represented byfold-change in phosphorylated NF-κB p65immunopositive cells in each ofthe EDC treatments compared to the vehicle control-treated iFGEs (***p<0.001) and iHTNs (*** p<0.001). Representative immunoblots for proteinlevels in whole cell lysate showing increases in phosphorylated p65,total p50 and total p52 levels in (FIG. 16B) iFGE, *** p<0.001 and (c)iHTNs *** p<0.001. Quantified histograms using ImageJ-based densitometryof bands for each of the respective immunoblots are shown below andrepresented as fold-change compared to vehicle-treated control. Ratio ofphosphorylated NF-κB p65 over total p65, p50/105 (canonical) andp52/p100 (non-canonical) were calculated. All statistical analysis wereperformed using one-way ANOVA. Images and data shown are representativeof average results from the two iPSC lines differentiated n=3 times inindependent experiments.

As shown in FIG. 17A-G: EDCs Induce Metabolic Stress and DisruptEndocrine Regulation. (FIG. 17A) immunoblots showing exemplary decreasesin phosphorylated p53 (Ser15) in both iFGE and iHTN (*** p<0.001) uponEDC exposure, (FIG. 17B) Seahorse mitochondrial respirometrymeasurements of with histograms representing changes in sparerespiratory capacity in iFGE and iHTN, * p<0.05; **p<0.01; (FIG. 17C)RT-qPCR relative normalized expression of nuclear (SCO2, POLRMT, TFAM)and mitochondrial--encoded (CYB5A) genes involved in mitochondrialrespiration from iHTNs. (FIG. 17D) Putative binding motifs for NF-78 Bp65 (RelA) and p53 transcription factors on the DNA of SCO2, POLRMT,TFAM, CYB5A, TP53, and RELA genes shown in the table displays number ofpossible binding sites and distance from transcription start site at aconfidence level of 70%; Red fonts IL1A and CDKN1A are known to bepositively regulated genes by p65 and p53 respectively, (FIG. 17E)Measurement of ATP levels (ATP/ADP ratio) showing decreases withEDC-treatments, (FIG. 17F) Immunoblots showing decreases in PYYlevels inEDCs treated iFGEs; (FIG. 17G) ELISA of α-MSH showing decreases insecretion with EDC treatment of iHTNs. * p<0.05, ** p<0.01, *** p<0.001,n=3. ND: Not detectable. All statistical analysis was performed usingone-way ANOVA. Data shown are representative of average results from thetwo iPSC lines differentiated n=3 times in independent experiments.

FIG. 18A-FIG. 18H shows that Blocking NF-kB Rescues EDC-mediatedMetabolic Stress & Endocrine Dysfunction. More specifically, immunoblotsshowing exemplary NF-kBi treatment decreases EDC-mediated increases inphosphorylated p65, p50, and p52 in (FIG. 18A) iFGEs and (FIG. 18B)iHTNs, *p<0.05, **p<0.01, *** p<0.001. Two different cell lines wereloaded in 6 lanes with lanes 1, 2 and 3 belonging to80iCTR (Vh1, Comb1and NF-κBi1) and lanes 4, 5 and 6 from 201iCTR (Vh2, Comb2 and NF-κBi2).(FIG. 18C) Immunocytochemistry showing phosphorylated p65 staining invehicle treatment (Vh), increased phospho-p65 with EDC combinationtreatment (Comb) that decreases with NF-κBi, * p<0.05, **p<0.01, ***p<0.001. (FIG. 18D) Seahorse assay showing improved mitochondrialrespiration upon NF-κBi treatment compared to combination treatment iniHTNs, *** p<0.001. (FIG. 18E) RT-qPCR expression levels of SCO2,POLRMT, TFAM and CYB5A showing decreased mitochondrial respiratory geneswith combination treatment that are rescued by NF-κBi treatment, *p<0.05, ** p<0.01, ***p<0.001. (FIG. 18F) Restoration of ATP levels uponNF-κBi treatment, **p<0.01, ***p<0.001; (FIG. 18G) α-MSH secretionlevels showed improvement upon NF-κBi treatment, ***p<0.001, (FIG. 18H)Western blot showing rescue of PYY levels in iFGEs, * p<0.05, **p<0.01.All statistical analysis was performed using one-way ANOVA. Images anddata shown are representative of average results from the two iPSC linesdifferentiated n=3 times in independent experiments.

FIG. 19 shows an exemplary schematic diagram of a cell showing aproposed model of EDC-mediated dysregulation in developing pluripotentstem cell-derived endocrine tissues. Developing endocrine cells whenexposed to EDCs such as PFOA, TBT and BHT trigger endoplasmic reticulum(ER) stress by increasing IRE1 and downregulation of Ero1 and BiP, whichare known to induce an unfolded protein response (UPR) in a cell. Thisresults in perturbation of NF-κB (increased phosphorylation of p65) andp53 (decreased phosphorylation of p53 at Ser15) signaling in parallel.The subsequent metabolic stress is comprised of reduced transcription ofboth nuclear- and mitochondrial-encoded respiratory genes, defectivemaximal respiration and mitochondrial spare respiratory, and a decreasein cellular bioenergetics/ATP levels. Intricate crosstalk betweenunhealthy mitochondria and ER may further lead to ER stress in afeedback loop and thereby exacerbate this mechanism. Overall, bothaccumulations of misfolded proteins as well as a decrease in ATP levelsupon chronic exposure to low-dose of EDCs induces metabolic stress in anendocrine cell, thereby negatively impacting endocrine regulation due toabnormal production and secretion of gut and brain neuropeptides.

EXAMPLE 22 Bioinformatics

Bioinformatic determination of putative DNA binding sites for NFκB-p65(RELA) and TP53 are shown in FIG. 20A-D. (FIG. 20A) Charts showingidentification of the number of putative binding sites of NFκB-p65 andTP53 binding motifs on genes of interest such as SCO2, POLRMT, TFAM,CYB5A and respective known genes regulated by NFKB-p65 (RELA) such asIL1A, IL1B, TNF, IL6 or regulated by TP53 such as GADD45A, GADD45B,GADD45G, PERP, BAX. (FIG. 20B) Identification of minimum distance inbase pairs upstream of the transcription start sites of the DNA bindingmotifs of NFκB-p65 and TP53 on the indicated genes of interest. HOXgenes were employed as neutral genes or genes that are not well-known inthe literature to be controlled either by NFκB-p65 and TP53. The DNAbinding motif as a sequence logo graphical representation of thesequence conservation of nucleotides where the size of the nucleotideletter represents the frquecny of the letter at that position in thesequence for (FIG. 20C) NFKB-p65 and (FIG. 20D) TP53 used in thebioinformatic analyses.

EXAMPLE 23 Developing a Stomach (Forgut) Microfluidic Chip

In this example, exemplary materials, cells and methods are describedfor developing a Foregut/stomach-chip for use, in part, as a human modelfor developmental effects of test agents and drugs. In otherembodiments, derived stomach cells, from foregut cells, are used fortesting agents and drugs.

Thus, a stomach cell differentiation protocol was developed herein fordifferentiating endoderm into foregut cells for further differentiatinginto stomach cells. FIG. 21 shows an exemplary stomach (foregut)optimization protocol for deriving cells to use on chips. A schematictimeline showing exemplary 3D organoid maturation from endoderm for anexemplary Foregut—stomach differentiation protocol. For example, cellsare iFG-O=iPSC-derived foregut organoids; iFG-O-diss=Day 34 organoidsdissociated; iFG-MO=Day 6 mini organoids and Epi-iFG=Day 6 miniorganoids sorted on Day 20.

For initial optimization experiments, Day 34 organoids were dissociatedinto single cells for use with chips. However, dissociation was harsh oncells and we did not get good cell survival on chips. Therefore, othercells were tested. FIGS. 22A-22I shows exemplary characterization of D34iFG-O by ICC. Fluorescent micrographs of cells and tissues stained withimmunomarkers for characterization of the cells/tissues used for seedingchips. Examples of markers, includig E-cadherin, Sox2, Muc5AC,synaptophysin, serotonin, somatostatin, gastrin, ghrelin, and peptideYY. Tissue stained in these micrographs shown are D34 are inducedorganoids.

Thus, in another embodiment, during the development of the presentinventions, Day 6 cells are plated on 3-D matrigel bubbles for 34 daysto obtain foregut organoids. These are dissociated into a monolayer andplated onto a chip as iFG-O-dins. In another embodiment, D60 (day 6organoids) in 2D culture are cultured for additional time, up to day 20.Then on Day 20, the cultures are flow sorted 2D for epithelial cells,e.g. Epi-iFG. In another embodiment, Day 6 cells are directly seeded as6-day mini organoids for obtaining iFG-MO. FIG. 23 shows an exemplaryoverall plan for cells to be used for seeding foregut on a chip. Aschematic timeline showing endoderm induction and foregutdifferentiation of iPSCs within increasing amounts of fetal bovine serum(FBS) in the presence of Activin A and Wnt3A followed by the addition ofCHIR, FGF4, LDN, and RA at day 3 onwards. iFG-O-diss=Day 34 organoidsdissociated; iFG-MO=Day 6 mini organoids; Epi-iFG=Day 6 mini organoidssorted on Day 20. Using flow sorted epithelial cells we believe will bea more streamlined approach to look at behavior of foregut on a chip. Wenamed the 3D dissociated organoid cells as iFG-O-diss, Day 6 miniorganoids as iFG-MO and the sorted cells are called Epi-iFG.

For cell type and ECM optimization, whole Day 6 spheroids was used forseeding chips. After several types of tests, optimizing ECM conditions,a 1:1 Laminin:Fibronectin for ECM coating was chosen for chips intendedto grow foregut cells. For Day 6 spheroids an applied 30 uL flow rateshowed more SYP positive cells vs no flow chips.

However, the 30 uL flow caused organoids to excessively grow. Also therewere high Sox2+ cells, indicating cells remained in progenitor stageinstead of maturing as desired, as we need more mature cell types.

Therefore, addition experimental results in this example show furthertweaking by decreasing flow to 10 uL to control excessive cellproliferation.

FIG. 24 shows an exemplary stomach-hypothalamus co-culture on a chip. Anexemplary schematic of one embodiment of a microchip. This chip showsiFG-MO cells in the upper channel with iHTN in the lower channel. Goal:To test if the presence of hypothalamic neurons (iHTNs) can beco-cultured on a chip. Approach: Apical channel was seeded with iFG-MOand the basal channel with iHTNs. Co-culturing foregut with iFG-MO (mo:minoorganoids) with induced hypothalamic neurons (iHTNs). We alsodecreased flow rate to 10 uL/hr due to over proliferation of iFG-MO inthe previous set of experiments.

FIG. 25A-D shows exemplary confocal microscopy images of fluorescingmarkers. Exemplary immunofluorescent micrographs of cells on chipsstained with immunofluorecent markers in upper and lower channels ofchips. FIG. 25A) All fluorescent channels showing immunofluorescenceemitting from upper and lower channels of the chip. FIG. 25B) Sox2fluorescence observed on apical region. FIG. 25C) E-cadherinfluorescence observed on apical region. FIG. 25D) TuJ1 fluorescenceobserved on basal region. Images showing markers in respective channelsand regions (see previous Figure for exemplary cells in upper and lowerchannels) under flow (10 ul/hr). The markers were very specific and werefound only in their respective channels.

This example describes an exemplary chip set up comparing no flow andflow conditions, e.g. (Flow 30 uL/hr). Chip set up: iPSC derived Stomachorganoids and iFG-MO were seeded to the apical channel; no cells wereseeded on basal channel for functional assay and imaging. Conditionsthat are monitored included but were not limited to functional assay andimaging for seeding efficiency on laminin/Fibronectin, imaging forforegut markers such as Sox2, E-cadherin and endocrine cells e.g.Synaptophysin.

Results of growing iFG-MO under flow. Flow still caused a lot of cellgrowth. More cells were maintained in progenitor stage than mature stage(Sox2+). Compared to no flow iFG-MO: Fewer Sox2+ and no continuousepithelium. Results of growing iHTN under flow. The cells didn't lookmorphologically great under flow conditions. Compared to no flow iHTNwhich showed relatively normal morphology.

FIG. 26A-C shows confocal imaging of IFG-MO on Day 21 under flow (30ul/hr). Exemplary immunofluorescent micrographs of cells in chipsstained with immunofluorecent markers. FIG. 26A) Foregut progenitorcells stained with DPAI and SOX2. FIG. 26B) Endocrine cells stained withSYP. And FIG. 26C) Epithelium stained with E-cadherin. FIG. 27A-B:iFG-MO seeded on apical channel. Flow (10 ul/hr). Exemplaryimmunofluorescent micrographs of cells in chips stained withimmunofluorecent markers. FIG. 27A) Fewer Sox2+ and FIG. 27B) Highernumbers of SYP+ cells in comparison to cells grown under 30 ul/hr flowrates. Results shows that iFG-MO cells on an apical channel under 10ul/hr flow conditions showed better epithelium coverage, (although inpatches instead of a continuous layer, slightly more SYP+ cells andhigher Sox2+ cells compared to no flow comparative chips. Cells grownunder this flow condition showed excessive cell growth compared to noflow cells.

Therefore, there was still excessive growth of, i.e. more Sox2+ cells offoregut organoids than desired; so further experiments were done asdescribed below and herein.

EXAMPLE 24 Maturing Foregut Cells and Hormone Effects

In some embodiments, maturing foregut cells were tested for effects ofchanging EGF levels on maturation, in part because of the relatively lownumbers of SYP+ cells under flow. In particular, alongside 10 uL flow(see the previous Example), EGF levels were decreased from 100 ng/mlinitially gradually to 2 ng/ml with one intermediate step of 10 ng/ml.See, FIG. 30. In part this was done to check if this made maturation toendocrine cells types better. This condition indeed showed fewer Sox2+cells and higher SYP+ cells under flow condition. But we did not getcomplete coverage of epithelium which was rather in patches.

At this point the selection of Day 6 organoids came out to be a crucialstep in obtaining good epithelium, based on some experiments performedin the lab and hence we tried a selection reagent which effectivelyseparate cell clusters from the surrounding monolayer and appeared to bean effective way to pick Day 6 organoids for plating.

Thus, in some embodiments, streamlining the picking of Day 6 organoidswas done. In order to get less of other cells types in the chip and getmore epithelium, we optimized, e.g. changed, the organoid selection stepby using a selection reagent instead of hand picking. In this attempt ofusing Selection reagent the foregut cells formed continuous epithelium

An exemplary selection reagent was used herein, e.g. STEMdiff™ NeuralRosette Selection Reagent, an Enzyme-free reagent for the selectivedetachment of neural rosettes. STEMCELL Technologies Inc. Catalog#05832.

FIG. 28 shows exemplary optimizing foregut epithelium. An exemplaryschematic of one embodiment of a microchip along with a schematictimeline for foregut and organoid maturation. Goal: To optimize theformation of foregut epithelium by better more streamlined selection ofDay 6 organoids using a Selection reagent. Approach: Apical channelseeded with iFG-SR by selecting organoids using a selection reagent.Maintained decreased flow rate at 10 uL/hr. EGF concentration wasdecreased in medium gradually over time to encourage differentiation andmaturation.

FIG. 29 shows exemplary experimental Timecourse showing lowering amountsof an agent. A schematic timeline showing iFG-SR cells grown underdecreasing amounts of a maturation agent, e.g. EGF.

This example describes an exemplary chip set up comparing no flow andflow conditions, e.g. (Flow rate 10 uL/hr) for iFG-SR cells compared toiFG-MO cells grown under flow movements.

Chip set up: iPSC derived Stomach organoids, iFG-MO, or iFG-SR cellswere seeded to the apical channel; no cells were seeded on basal channelfor functional assay and imaging. Conditions that are monitored includedbut were not limited to functional assay and imaging for foregut andendocrine markers such as Sox2, E-cadherin and Synaptophysin, inaddition to Measuring hormone secretion levels (Ghrelin) using ELISA

FIG. 30 shows exemplary general characterization of the tissue used forseeding chips. Exemplary immunofluorescent micrographs of cells on chipsstained with immunofluorecent markers, e.g. E-cadherin, Sox2, Sox17,synaptophysin, serotonin, somatostatin, gastrin, ghrelin, and peptideYY. Characterization of D20 iFG-SR cells by ICC on a 96-well plate (2DDay 20).

FIG. 31A-B shows exemplary comparative tile scan images of iFG-SR andiFG-MO stained for E-cadherin. Exemplary immunofluorescent micrographsof cells on chips stained with an immunofluorecent marker forE-cadherin. FIG. 31A) iFG-SR and FIG. 31B) iFG-MO. Under flow rate of 10ul/hr.

FIG. 32 shows exemplary Ghrelin secretion by ELISA assay comparing SRand hand picked D6 organoids (O). Several exemplary cultures of iFG-SRand iFG-MO were compared for ghrelin secretion (pg/mg of cell protein)from day 15-22 and day 23-30 of chip culture.

In this attempt of using a selection reagent the iFG-SR formedcontinuous epithelium. iFG-SR Flow conditions showed higher numbers ofSYP+ cells compared to iFG-MO (flow) and iFG-SR (no flow movements). Wealso observed detectable levels of Ghrelin secreted by these stomachcells, which increased over time with flow growth conditions.

EXAMPLE 25 Hormone Effects and Cancer

We used a human gastric cancer cell line as a positive control tocompare secretion capabilities of our organoids (O). We obtainedendocrine cells as seen both by staining (SYP) and ELISA (Ghrelin). Wewere able to control over proliferation by controlling EGF levels. Wewere also able to increase maturation of endocrine cells by controllingEGF levels. Ghrelin secretion levels of iFG-SR were observed comparableto HGC secretion levels.

This example describes an exemplary chip set up and comparison betweenno flow and flow conditions, e.g. (Flow rate at 10 uL/hr) comparingiFG-SR and HGC cells.

For iFG-SR cells, growth conditions included: Day 1: Seed iFG-SR. Day 3:Start flow movement on chips using 10 ul/hr at 100 ng/ml EGF. Day 11:Lower EGF to 10 ng/ml. Day 14: Further lower EGF to 2 ng/ml. Day 21 Stopexperiment.

For HGC, growth conditions included: Day 1: Seed HGC. Day 3: Start flowmovement on chips of 10 ul/hr at 100 ng/ml EGF. Day 21: Stop experiment.

Chip set up: iFG-SR or HGC were seeded to the apical channel; no cellswere seeded on basal channel for functional assay and imaging.Conditions that are monitored included but were not limited tofunctional assay and imaging for foregut and endocrine markers such asSox2, E-cadherin and Synaptophysin (SYN), in addition to measuringhormone secretion levels (Ghrelin) using ELISA. FIG. 33 shows anexemplary schematic of one embodiment of a microchip along with aschematic timeline for foregut and organoid maturation including aselection reagent and decreasing amounts of EGF. In this exemplaryexperimental set up methods of culturing are described for comparison ofthe foregut system, as described herein, with a positive control(NCI-N87 gastric cancer line). Goal: To compare iFG-SR to human gastriccancer (HGC) (NCI-N87-epithelial) line. Approach: Apical channel seededwith iFG-SR or HGC. Maintain decreased flow rate at 10 uL/hr. Comparethe 2 cell types on chips by ICC and Ghrelin secretion. The HGC line ismaintained in their optimal growth medium with no variations throughoutthe experiment.

At this point the selection of Day 6 organoids came out to be a crucialstep in obtaining good epithelium, based on some experiments performedin the lab and hence we tried a selection reagent which effectivelyseparate cell clusters from the surrounding monolayer and appeared to bean effective way to pick Day 6 organoids for plating, see herein andabove.

FIG. 34A-B shows exemplary flow condition effects on HGC and iFG-SRcells in chips as micrographs of cell layers comparing SOX2, SYP andE-cadherin (E-cad) immunofluorescent staining between FIG. 34A) HGC andFIG. 34B) iFG-SR cells. FIG. 35 shows a comparative tile scan of HGC andiFG-SR cell layers as exemplary comparative micrographs of cell layerscomparing iRG-SR and HGC growing with and without flow conditions inchips. Flow worked better for iFG-SR but not for HGC. iFG-SR epitheliumlooked better under no flow conditions than under flow movement.

FIG. 36 shows an exemplary steady increase in Ghrelin secretion withflow movement in iFG-SR chips compared to lower amounts secreted fromiFG-SR cells in no flow chips.

Therefore, we obtained endocrine cells as seen both by ICC (SYP) andELISA (Ghrelin). We were able to control over proliferation bycontrolling EGF levels. Further, we were also able to increasematuration of endocrine cells by controlling EGF levels.

EXAMPLE 26 Exemplary Experimental Flowchart and Set Up

This example describes an exemplary chip set up and comparison betweenno flow and flow conditions, e.g. (Flow rate at 10 uL/hr). Chip set up:iPSC derived Stomach organoids and iFG-MO seeded to apical channel;iHTNs seeded on basal channel for functional assay and imaging.Conditions that are monitored included but were not limited to, growthof iHTNs in chip and imaging for foregut and neuronal markers such asSox2, E-cadherin and TuJ1. See, FIG. 37, Exemplary experimentalflowchart and set up. A schematic timeline showing an exemplary chip,experimental conditions and examples of assays. iPSC derived Stomachorganoids and iFG-MO seeded to the apical channel; iHTNs seeded on thebasal channel for functional assay and imaging; growth of iHTNs in chipand imaging for foregut and neuronal markers such as Sox2, E-cadherinand TuJ1. Cultured in duplicate under no flow and flow conditions (Flow10 uL/hr).

EXAMPLE 27 General iPSC Reprogramming Protocol for Lymphoblastoid CellLine

Disease modeling can benefit greatly from using patient specific stemcells to recapitulate disease features, allowing observation ofdevelopmental features. A significant resource for iPSC generationincludes lymphoblastoid cell lines, for which a variety of worldwiderepositories exist. An improved method for reprogramming from thesesources, can be described as first involving nuclection of a target hostcell with a combination of plasmids, followed by 2 days of incubation,daily addition of reprogramming media (without aspiration of old media)on each of days 3-5, replacement of reprogramming media (withaspiration) on day 6, daily addition of reprogramming media (withoutaspiration of old media) on each of days 7-9, replacement ofreprogramming media (with aspiration) on day 10, alternate dailyaddition of reprogramming media (without aspiration of old media) ondays 10-16, Small colonies may appear as early as day 11, withsubstantial numbers of colonies becoming visible by day 17. Mediaswitching into progressively increasing amounts of serum-free, completemedia, mTeSR1 is provided on days 18-20. By day 24, reprogrammedcolonies are readily apparent, and can be antibody stained for live cellimaging for confirmation. Throughout days 25-29, additional colonies canbe isolated for sub-cloning. By day 30, previously isolated coloniesbegin to adhere, display normal iPSC morphology and can be stored orsubsequently serially passaged as cell lines. Using the describedtechniques the inventors can achieved at least 10% conversionefficiency, representing at least 3-8 fold improvement compared toexisting reprogramming studies. Additional details are found in PCT App.No. PCT/US2015/034532, which is fully incorporated by reference herein.

EXAMPLE 28 Three-Dimensional Intestinal Organoids and IntestinalEpithelial Cells From iPSCs

To induce definitive endoderm formation, all iPSCs were cultured with ahigh dose of Activin A (100 ng/ml, R&D Systems) with increasingconcentrations of FBS over time (0%, 0.2% and 2% on days 1, 2 and 3respectively). Wnt3A (25 ng/ml, R&D Systems) was also added on the firstday of endoderm differentiation. To induce hindgut formation, cells werecultured in Advanced DMEM/F12 with 2% FBS along with Wnt3A and FGF4 (500ng/ml, R&D Systems). After 3-4 days, free-floating epithelial spheresand loosely attached epithelial tubes became visible and were harvested.These epithelial structures were subsequently suspended in Matrigelcontaining R-Spondin-1, noggin, EGF (500 ng/ml, 100 ng/ml and 100 ng/mlrespectively, all R&D Systems) and then overlaid in intestinal mediumcontaining R-Spondin-1, noggin, EGF (500 ng/ml, 100 ng/ml and 100 ng/mlrespectively, all R&D Systems) and B27 (1X, Invitrogen). Organoids werepassaged every 7-10 days thereafter.

EXAMPLE 29 Seeding of Intestinal Epithelial Cells into the MicrofluidicDevice

To seed intestinal epithelial cells into the microfluidic device, HIOswere first dissociated and the intestinal epithelial cells were thenobtained using fluorescent activated cell sorting. 24 hours prior tosorting, ROCK inhibitor (10 μM, Tocris) was added to HIO culture media.The following day, HIOs were removed from Matrigel and subsequentlyincubated in TrypLE Express (Life Technologies) for between 20-40 minuntil the organoids are completely disassociated to a single cellsuspension. These cells were then passed through a 30 micrometer filterand stained with CD326 (Biolegend) for 30 min. Cells were thenpositively sorted for CD326. Cells were collected and resuspened to adensity of 5×10⁶/ml in intestinal media containing ROCK inhibitor (10μM, Tocris), SB202190 (10 μM, Tocris) and A83-01 (500 nM, Tocris).Dead/non-adhered cells were removed after 3-6 hours by flushing mediathrough the device and flow was started 8-24 hrs later at a rate 60ul/hr.

EXAMPLE 30 Seeding of Intestinal Epithelial Cells into the MicrofluidicDevice

Intestinal epithelium, derived from iPSCs, is seeded onto themicrofluidic device followed by characterization of intestinalepithelial subtypes. Functional assays, including an examination ofpermeability via transepithelial resistance and dextran FITC efflux willbe assessed either under basal conditions or in response to inflammatorycytokines such as interferon-gamma (IFNg) and/or tumor necrosisfactor-alpha (TNFalpha). Also drug candidates that may modulate thevarious intestinal epithelial subtypes will be examined to assess ifsuch subtypes can indeed be modulated. After establishing such assays,IPSCs from genetically defined inflammatory bowel disease (IBD) patientswill be generated, differentiated into intestinal organoids,disassociated and subsequently seeded onto the microfluidic devices andthe functional consequences of the genetic variations associated withIBD will be assessed.

EXAMPLE 31 Obesity Model

This example (and the next) are directed to cells associated with anobesity model. Non-integrating iPSC lines were generated fromindividuals with normal body mass index (BMI<25) and super obese (SO)with BMI>50. Feasibility was shown for iPSC re-differentiation intoendocrine tissues—gastrointestinal (GI) organoids and hypothalamic (HT)neuropeptidergic neurons. Differential baseline whole cell proteomeprofiles were generated from their iPSC-endocrine cells. Differentiationof iPSCs to gastrointestinal organoids (iGIOs) and hypothalamic neurons(iHTNs) was done in advance of seeding cells on “organ-on-chip”microfluidic devices. An exemplary microfluidic device contemplated foruse is shown in FIG. 38A-B with exemplary results of using iGIOs andiHTNs on chips shown in FIG. 39A-G.

EXAMPLE 32 Chronic Low Dose Treatments of Microfluidic “Organ-on-Chip”Devices with EDCs

We hypothesize that chronic low-dose exposure to endocrine disruptingchemicals (EDCs), is deleterious during early human endocrine tissuedevelopment, resulting in hyperactive NF-κB and HMG proteinpro-inflammatory signaling with permanent mitochondrial dysfunction. Totest this, the gastrointestinal organoids (iGIOs) and hypothalamicneurons (iHTNs) seeded on “organ-on-chip” microfluidic devices (Example31) are exposed to chronic low-dose treatments (TDI range) of EDCpollutants/mixtures (e.g. tributyltin (TBT), perfluorooctanoic acid(PFOA), butylated hydroxytoluene (BHT), and bis(2-ethylhexyl) phthalate(DEHP); dysregulated secreted protein groups will be identified byquantitative proteomics.

EXAMPLE 33 Microfluidic “Organ-on-Chip” Devices Seeded with Single CellSuspensions

In one embodiment, iPSCs were directed to form HIOs and weresubsequently dissociated to a single cell suspension. These cells werethen seeded into a small microfluidic device (SMD) which is composed oftwo chambers separated by a porous flexible membrane. See, FIG. 40.

The presence of Paneth cells, goblet cells, enteroendocrine cells andenterocytes in these structures was confirmed by immunocytochemistrywhile in situ hybridization revealed the presence of Igr5+ cells.

Secretion of antimicrobials from Paneth cells was detected by ELISA andadministration of IFNgamma to the lower channel resulted in thephosphorylation of STAT1 and significant upregulation of IFNgammaresponsive genes including, but not limited to, IDO1, GBP4 and/or GBP5.Interestingly, phospholipase A2 group 2A and Muc4, two genes specific tointestinal epithelial subtypes, were also upregulated. When compared toCaco2 cells, there was no corresponding upregulation of genes associatedwith these epithelial subtypes.

EXAMPLE 34 Microfluidic “Organ-on-Chip” Devices Compared to TranswellCulture Devices

iGIOs and iHTNs were seeded in both dynamic flow microfluidic devises,FIG. 38A, and static trans-well devices, FIG. 38B. Exemplary FIG. 38A-Bshows exemplary seeding for EDC perturbation of iGIOs (apical) and iHTNs(basal) as dynamic flow organs-on-chips (Dynamic flow OoC) and statictranswell culture. These systems were tested (+/− (control) withexemplary compounds including but not limited to TNF-alpha and EDCs.Dynamic flow OoC: PDMS membrane has 7 um pores. Apical channel is 1 mmhigh while the basal channel is 0.2 mm high.

In addition to differences in media flow, these devices have inversedorientations of cells. For example, culture devices used for testingcompounds on iGIOs and iHTNs cells under flow microfluidic devices areapical and basal, respectively. However in the static trans-wells thesecells are instead iGIOs (basal) and iHTNs (apical).

FIG. 38A-B: One embodiment of an “Organ on chip” microfluidic device. Anexemplary schematic diagram illustrating the difference between statictranswell culture of gastrointestinal organoids (iGIOs) and hypothalamicneurons (iHTNs), which were differentiated from iPSCs, and culture underflow conditions in “organ on chip” microfluidic devices.

FIG. 39A-G: Exemplary Results Using An “Organ on chip” MicrofluidicDevice Of The Previous Figure. Provides exemplary experimental resultsof immunostaining of cells using an organs-on-a-chip model of iGIOs andiHTNs. FIG. 39A) shows a chip with apical (Red) and basal (Blue)channels. FIG. 39B) shows a micrograph of iGIOs differentiated on theapical channel. FIG. 39C) shows GI epithelium on chip that isE-cadherin+(white) with Sox2+ foregut progenitors (green). FIG. 39D)shows iGIOs on chip showing epithelium as E-cadherin+(white) andsynaptophysin+ (SYN) endocrine cells (red). FIG. 39E) shows a confocal3D image of seeded chip with iHTNs in the basal channel (orange TuJ1+),while FIG. 39F and FIG. 39G show Sox2+foregut, and E-cadherin+epitheliumin apical channels only (respectively). White arrows point to the porousmembrane while * identifies a lumen surrounded by neuronal cells in FIG.39E-FIG. 39F.

FIG. 40 shows an illustrative schematic of one embodiment of a smallmicrofluidic device illustrating upper and lower chambers separated by aporous membrane. Arrows represent continuous flow of media in both upperand lower channels. Vacumm chambers are located on the outside of bothsides of the channel areas.

EXAMPLE 35 Epithelial Cells in Microfluidic Cultures

Human intestinal epithelial cells derived from IPSCs were treated with10 ng/ml of IFNgamma. The basal administration of IFNg leads to adecrease in transepithelial resistance and an increase in the efflux ofdextran FITC in human intestinal epithelial cells derived from IPSCs.Basically this means that the intestinal epithelium is more permeable inresponse to this cytokine. The addition of TNFa does not elicit anychange in intestinal permeability.

IFNgamma treatment resulted in a loss of transepithelial electricalresistance (TEER) over time as shown in the graph in FIG. 43A. Control(untreated) and TNFalpha treated cells showed increased TEER over timecomparable to controls FIG. 43A. n=4. Similarly when FITC dextrin addedto the apical channel INFgamma treatment caused an increase inpermeability co-efficient, FIG. 43B, and accumulation in the basallayer, FIG. 43C. TNFalpha treated cells and control cells showedcomparable apparent permeability co-efficients and basla accumulation ofFITC dextran.

FIG. 43A-G: Shows exemplary graphs demonstrating IFNgamma effects onhuman intestinal epithelial cells derived from IPSCs in microfluidicchips. Graphs show a loss of electrical resistance (TEER) and a loss ofconnections between epithelial cells treated with IFNgamma. FIG. 43A)TEER was reduced over time with IFNgamma treatment while control andTNFalpha treated cells showed increased TEER. FIG. 43B) FITC dextrinadded to the apical channel showed a similar loss as permeabilityco-efficients, and FIG. 43C) showed increased amounts of FITC dextrin inthe basal layer (after addition to the apical layer) for IFNgammatreated cells.

EXAMPLE 36 Three Dimensional Organoid System Developed for use in aMicrofluidic “Organ-On-Chip” Device

We grew intestinal organoids, e.g. shown in FIG. 41, that have all ofthe cell types typically found in the intestine. As examples, individualcells are shown fluorescently stained in micrographs of FIG. 42A-D.These include enterocytes involved with nutrient absorption, Gobletcells involved with producing mucus, Paneth cells involved withproducing anti-microbial agents, and enteroendocrine cells involved withproducing hormones.

Further, propagation of a three dimensional organoid system iscontemplated for use in: analysis of cytokines on the host side;analysis of epithelial subtypes; permeability; apical administration ofpeptides; bacterial interactions; and co-culture with immune cells.

FIG. 42A-D: Shows fluorescently stained micrographs of intestinalorganoid cells. FIG. 42A) enterocyte, tissue stained with Caudal TypeHomeobox 2 (CDX2) and Fatty Acid Binding Protein 2 (FABP2); FIG. 42B)Goblet cells, tissue stained with CDX2 and Mucin 2 (MUC2); FIG. 42C)Paneth cells, tissue stained with CDX2 and lysozyme; and FIG. 42D)enteroendocrine cells, tissue stained with CDX2 and Chromatogranin A(parathyroid secretory protein 1), typically located in located insecretory vesicles.

EXAMPLE 37 Microfluidic “Organ-on-Chip” Device

Exemplary schematics and cells growing on microfluidic chips are shownin FIG. 44A-E. FIG. 44A) Shows schematic illustration of chip; FIG. 44Band FIG. 44C) shows photographs with overlays identifying parts andsizes of a “Gut On A Chip”; FIG. 44C) additionally shows a micrograph ofthe membrane; FIG. 44D) Shows schematic illustration of a chip withoutand with mechanical strain with micrographs of resulting cells beloweach representation; and FIG. 44E) shows a graph of substrate strain (%)vs. cell strain (%) in relation to applied pressure (kPa).

Examples of seeded channels were fluorescently stained to show cells.Examples of stains show FIG. 45A) with DAPI (nuclei), FIG. 45B)E-cadherin, with an overlap of the two fluorescent channels shown inFIG. 45C.

A comparison of cells cultured with and without media flow show thatflow conditions produce a continuous coverage of cells, unlike the cellsgrown without flow. FIG. 46 shows Cells cultured under static conditionsfor 6 days while FIG. 47 shows cells cultured under flow conditions for6 days.

FIG. 44A-E: Shows Exemplary “Gut On A Chip” Technology. FIG. 44A) Showsschematic illustration of chip; FIG. 44B and FIG. 44C) shows photographswith overlays identifying parts and sizes of a “Gut On A Chip”; FIG.44C) additionally shows a micrograph of the membrane; FIG. 44D) Showsschematic illustration of a chip, without and with mechanical strain,with micrographs of resulting cells below each representation; and FIG.44E) shows a graph of substrate strain (%) vs. cell strain (%) inrelation to applied pressure (kPa).

FIG. 45A-C: Shows Epithelial Cells Growing in Channels of a “Gut On AChip”. Examples of seeded channels were fluorescently stained FIG. 45A)with DAPI (4′,6-diamidino-2-phenylindole), a fluorescent stain thatbinds strongly to A-T rich regions in DNA) (nuclei), FIG. 45B)E-cadherin, with an overlap of the two fluorescent channels shown inFIG. 45C).

FIG. 46: Shows exemplary cells cultured under static conditions for 6days in a microfluidic chip. Cells do not form a continuous layer.

FIG. 47: Shows exemplary cells cultured under flow conditions for 6 daysin a microfluidic chip. Cells form a continuous layer.

EXAMPLE 38 Caco-2 Epithelial Cells are Different than Enteroids whenGrown on Microfluidic “Organ-on-Chip” Devices

Caco-2 epithelial cells grown on chips do not show the same response toIFN-gamma as the enteroids grown on chips. In fact, a panel of markerscomparing relative expression of IFNgamma treated enteroids cells vs.Caco-2 epithelial cells with and without IFN-gamma showed differentresponses for each gene marker tested. FIG. 48A-I shows graphs ofrelative exemplary expression of gene markers normalized toGlyceraldehyde 3-phosphate dehydrogenase (GADPH) with and withoutIFNgamma treatment: FIG. 48A) IDO1 (indoleamine 2,3-dioxygenase 1); FIG.48B) GBP1 (guanylate binding protein 1); FIG. 48C) GBP4 (guanylatebinding protein 4); FIG. 48D) LYZ (Lysozyme); FIG. 48E) PLA2G2A(Phospholipase A2 Group IIA); FIG. 48F) a secreted antibacterial lectin(RegIIIγ); FIG. 48G) LRG5 (Leucine Rich Repeat Containing GProtein-Coupled Receptor 5); FIG. 48H) OLM4 (Olfactomedin 4); and FIG.481) MUC4 (Mucin 4).

Further, as sown in FIG. 48C, intestinal cells on microfluidic chipswith and without IFNgamma show more antibacterial lectin (RegIIIγ) theCaco2 cells regardless of whether they were treated with IFNgamma.

EXAMPLE 39 Spontaneous Formation of Polarized Intestinal Villous-LikeStructures in a Microfluidic “Organ-on-Chip” Device

Intestinal epithelial cells derived from human intestinal organoids weregrown in microfluidic chips as described herein. Twelve days afterseeding chips, cells were confluent with a continuous layer extendingpast the bend on the end of the upper channel of the chip. See, FIG. 49.

The chip device was then cut in cross section, as represented by the redline in FIG. 50 for viewing the chip and cells on end, similar to ahistological section view from a biopsy cut in a similar plane. A lightmicrograph of the cut axis through the chip shows the intestinal cellswith microvilous-like structures growing on the membrane in the upperchannel of the chip. For reference, the membrane, lower channel, andvacuum chambers are identified in FIG. 51.

For identification of cell types, cells were fluorescently stained formarkers and visualized in cross section, as represented by the red linein FIG. 50.

Surprisingly, cells grown under a continuous flow of media in both theupper and lower channels resulted in the spontaneous formation ofpolarized (e.g. apical and basal regions of cells) intestinalvillous-like structures that are similar to those found in vivo.

FIG. 52 represents an exemplary photomicrograph showing epithelial cellsderived from human intestinal organoids forming villous like structuresin response to a continuous flow of media in an upper and lower chamberof a small microfluidic device.

Immunofluorescence staining of a cross section was done to furtheridentify cells Double immunofluorescence staining of a cross sectionshows Caudal Type Homeobox 2 (CDX2) (red) and E-Cadherin (blue). Inaddition to Caudal-Type Homeobox Protein 2 (CDX2), a protein regulatorof intestinal gene expression typically found in the nucleus, andE-cadherin protein, a major component of adherens junctions attachingneighboring epithelial cells, staining for intestinal markers furtherincluded Intestinal-Type Fatty Acid-Binding Protein (FABP2), a cytosolicfatty acid transporter protein found in intestinal cells, and ZonaOccludens 1 (also Tight Junction Protein 1(TJP1)), a protein located ona cytoplasmic membrane surface of intercellular tight junctions. Tripleimminofluorsecence staining shows the presence of CDX2 (red) andE-Cadherin (blue) compared to FABP2 (green), FIG. 53, bar=100microm.Another triple imminofluorsecence staining shows the presence of CDX2(red) and E-Cadherin (blue) compared to ZO-1 (green), FIG. 54.

Thus, intestinal cells grown under flow in microfluidic chips from humanenteroids show intestinal 3D architecture mimicking human intestinaltissue. In part, microvilli are observed where CDX2 stained nucleisuggest a layer of epithelial cells folded into microvilli-likestructures.

Similar to human intestinal epithelial cells, these cells showcharacteristics of having intercellular attachments forming a barrierbetween the extracellular apical and basal regions. For example, theborders of two cells are typically fused together, often around thewhole perimeter of each cell, forming a continuous belt like junctionknown as a tight junction or zonula occludens (zonula=latin for belt).Other types of junctions include adherens junctions. The presence ofE-cadherin in addition to ZO-1, and physiological data showing TEERvalues indicative of barrier function support the observation thatintestinal cells grown on fluidic microchips are modeling humanintestinal linings.

EXAMPLE 40 Cell Seeding Density for Microfluidic Chip

This example shows exemplary results of seeding chips using differentamounts of cells in single cell suspensions of intestinal enteroids. Atleast 5 different chips were seeded with a range in amounts of cells per40 ul of fluid. Images of intestinal cells grown in microfluidic chipsseeded at densities 3.75×106 cells/mL (150K in 40 uL) and 2.5×10⁶cells/mL (100K in 40 uL), shown in FIG. 55D, Day 6 of incubation, andFIG. 55E, Day 7 of incubation were not seeded with enough cells. Highermagnified images of cells growing on top of the membrane in themicrofluidic chip also supported the lack of confluent coverage at thesecell numbers. For example, FIG. 56 shows that 3.75×10⁶ cells/mL (150K in40 uL) was not enough to provide a confluent coverage, see exemplarybare area outlined in red. FIG. 57 shows that 2.5×10⁶ cells/mL (100K in40 uL) was not enough to provide a confluent coverage, see severalexemplary bare areas outlined in red. In contrast, 7.5×10⁶ cells/mL(300K in 40 uL); 6.25×106 cells/mL (250K in 40 uL); and 5.0×10⁶ cells/mL(200K in 40 uL) were enough cells to provide a confluent layer of cells.

Amounts of cells ranged from 7.5×10⁶ cells/mL (300K in 40 uL)—2.5×10⁶cells/mL (100K in 40 uL). See, FIG. 55A-E. Confluent coverage wasobtained from ranges of cells from at least 300K down to at least 200Kand above 150K per chip. Nonconfluent coverage was observed from ranges150K-100K. See, red circled areas for nonconfluent coverage in FIG. 56and FIG. 57.

In one embodiment, a microfluidic chip disclosed herein is seeded with aspecified number of enteroid cells per channel, as a single cellsuspension, for providing a confluent coverage of the seeded channel. Inone embodiment, single cells suspensions of enteroids cells ranges fromabove 150K to 300K or more per chip.

FIG. 55A-E: Shows exemplary images taken after seeding chips. FIG. 55A)7.5×10⁶ cells/mL (300K in 40 uL); FIG. 55B) 6.25×10⁶ cells/mL (250K in40 uL); FIG. 55C) 5.0×10⁶ cells/mL (200K in 40 uL; FIG. 55D) 3.75×10⁶cells/mL (150K in 40 uL); and FIG. 55E) 2.5×106 cells/mL (100K in 40uL).

FIG. 56: Shows exemplary magnified images of nonconfluent areas afterseeding chips. Enteroid cells seeded at 3.75×10⁶ cell/mL (150K in 40 uL)(compare to FIG. 55D). Red circle outlines a nonconfluent area.

FIG. 57: Shows exemplary magnified images of nonconfluent areas afterseeding chips with fewer cells than previous image. Enteroid cellsseeded at 2.5×10⁶ cell/mL (100K in 40 uL) (compare to FIG. 55E). Redcircles outline nonconfluent areas.

EXAMPLE 41 Identifying Media Formulations for use in Apical and BasalChannels of Microfluidic Intestinal Organoid Chips

After identifying optimal culture time of organoids prior to use forseeding chips, e.g. age of organoids to seed chips, establishing rangesof organoid single cell suspension seeding density of the upper channel,and discovering that a flow rate of 30 ul/hour (in both channels)induces spontaneous formation of villous-like structures, mediaformulations were tested for identifying media resulting in viable cellcoverage of the upper channel of the microfluidic chip. In part, onegoal was to assess if media containing growth factors was required inboth the upper and lower channels for desired cell growth andcharacteristics. Exemplary media formulations are provided below in thisexample.

Single cells suspensions of intestinal organoid cells in complete mediawere seeded into an apical channel of the microchip then incubated for 4hours at 37 degree Celsius, after which a flow rate of 30 ul/hour wasapplied to the upper-apical and lower-basal channel of the chip alongwith the media described herein and shown in an exemplary schematicExperimental Design, FIG. 58. At least two types of media in at least 4combinations were tested in upper-apical cannels and lower-basalchannels: Complete (A)/Complete(B); GFR(A)/Complete(B);Complete(A)/GFR(B); and GFR(A)/GFR(B).

Exemplary complete (Complete) media: Advanced DMEM/F12 (Dulbecco'sModified Eagle Medium/Ham's F-12), L-Glutamine andPenicillin/Streptomycin (antibiotics) (1X), CHIR99021 (aminopyrimidinederivative: may be referred to as6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile)(2 mM), Noggin (glycoprotein: human recombinant) (100 ng/ml), EGF(Epidermal growth factor: human recombinant) (100 ng/ml) and B27 (serumfree supplement) (1X).

Exemplary growth factor reduced (GFR) media: Growth factor reduced mediais the following: Advanced DMEM/F12, L-Glutamine andPenicillin/Streptomycin (1X) and B27 (serum free supplement) (1X).

As shown in FIG. 59C on day 6 of culture, islands of intestinal cellsare observed that did not form a confluent layer when grown inComplete(A)/GFR(B) with even less coverage of the membrane observed inFIG. 59D GFR(A)/GFR(B). In contrast, as shown in FIG. 59A and FIG. 59B,a confluent coverage of cells over the membrane is obtained usingComplet (A)/Complete (B); GFR(A)/Complete(B), respectively. Compared toan additional day of culture, the use of both Complete (A)/Complete(B);GFR(A)/Complete(B) resulted in complete coverage of the membrane, FIG.60A and FIG. 60B, respectively, while a chip shown in FIG. 60C, Complete(A)/GFR (B), continues to show intestinal islands with incompletecoverage of the membrane. Direct comparisons betweenComplete(A)/Complete(B) vs. GFR(A)/Complete(B), while both showedconfluent coverage and villous-like structure at day 6 and 7, density ofvillous-like structures appeared higher with Complete(A)/Complete(B) vs.GFR(A)/Complete(B) in both FIG. 59A vs. FIG. 59B and FIG. 60A vs. FIG.60B.

This difference in growth was more apparent under growth conditions usedfor images shown in FIG. 61A-B where the use of Complete(A)/Complete(B)in FIG. 61A is clearly superior to use of GFR(A)/Complete(B) shown inFIG. 61B. Therefore, complete media containing the entire set of growthfactors in both channels results in superior growth and maintenance ofintestinal enteroid cells used in microfluidic chips of the presentinventions.

Thus, in one embodiment, complete media used in both upper (apical) andlower (basal) channels of a microfluidic chip disclosed herein. Use ofcomplete media results in growth of organoid cells providing a confluentcoverage and villous-like structures over the apical surface of themembrane in the upper channel.

FIG. 58: Shows exemplary schematic Experimental Design for media testingon cell growth. In part, this design is to determine whether mediacontaining complete growth factors should be used in both upper-apicaland lower-basal channels for growing intestinal enteroid cells in themicrofluidic chip.

FIG. 59A-D: Shows exemplary Day 6 magnified images of intestinalenteroid cells growing on chips comparing media formulations in upper(apical) and lower (basal) channels. Media comparisons are: FIG. 59A)Complete(A)/Complete(B); FIG. 59B) GFR(A)/ Complete(B); FIG. 59C)Complete(A)/GFR(B); and FIG. 59D) GFR(A)/GFR(B).

FIG. 60A-C: Shows exemplary Day 7 magnified images of intestinalenteroid cells growing on chips comparing media formulations in upper(apical) and lower (basal) channels. Media comparisons are: FIG. 60A)Complete(A)/Complete(B); FIG. 60B) GFR(A)/ Complete(B); and FIG. 60C)Complete(A)/GFR(B).

FIG. 61: Shows exemplary magnified images of intestinal enteroid cellsgrowing on chips showing growth differences between two mediaformulations inducing microvillous-like structures. Media comparisonsare: FIG. 61A) Complete(A)/Complete(B) and FIG. 61B) GFR(A)/Complete(B).

EXAMPLE 42 Flow Cytometric Analysis of Intestinal Cells Growing in aMicrofluidic Intestinal Organoid Chip

This example shows exemplary results of percentages of intestinal cellpopulations, derived from iPSC enteroids, growing in microfluidic chipsdescribed herein. The majority of cells grown in the microfluidic chipare epithelial cells (as exemplary 83.4% and 72% cell populations).Further, non-epithelial cell populations were identified in exemplarypopulations as 15.6% and 28.6% of the intestinal cells. Moreoverspecific non-epithelial cell types were also detected in an intestinalsmall cell population, including Paneth cells (5.03%), Enteroendocrinecells (0.153%), Goblet cells (0.131%), and Enterocytes (1.06%).

In brief, for flow cytometric analysis, intestinal cells were removedfrom chip membranes and processed for providing single cell suspensionsfor fluorescent antibody staining and flow cytometry analysis of cellpopulations. Cell populations were identified by forward scatter onscatter plots, i.e. FCS, for gating into populations for fluorescentanalysis.

Intestinal epithelial cells were identified with primary antibodiestargeting Epithelial Cell Adhesion Molecule (EpCAM) while nonepithelialcells were identified with Vimentin, a type III intermediate filament(IF) protein expressed in non-epithelial cells. Paneth cells,Enteroendocrine cells, Goblet cells, and Enterocytes were identifiedusing antibodies specific for each of those cell types.

Primary antibodies that were not directly conjugated with a fluorescentmolecule were indirectly detected using a secondary fluorescencatedantibody capable of binding to the primary antibody. Some antibodieshave background binding of their Fc region onto cells so that isotypecontrols were done for detecting background fluorescent binding of theantibody. Additionally, cells show varying amounts of autofluorescencewhen analyzed on certain fluorescent channels so that autofluorescenceof cells is used in part for setting fluorescent intensity gates (i.e.outlines shown in florescent dot plots).

FIG. 62A-F: Shows exemplary flow cytometry dot plots of enteroidiPS-derived intestinal cells as percentages of epithelial andnon-epithelial size gated cells from a microfluidic chip after 12 daysof incubation. FIG. 62A) Scatter plot showing intestinal cells sizegated as outlined at the flat end of the arrow into FIG. 62B) two-colorfluorescence dot plots showing background (auto) fluorescent intensityon two fluorescent channels and in *-fluorescent gated areas.Autofluorescence in gated areas for each fluorescent channel (*-outlinedfor fluorescent gating) shows 0.212% fluorescence (*-upper leftquadrant) and 0.004% (*-lower right quadrant) with a cell populationemitting autofluorescence on both channels shown in the populationgrouping in the lower left quadrant of the plot; FIG. 62C) Scatter plotshowing cells previously incubated with secondary fluorescent antibodyonly (another control for background) with cells gated as above for FIG.62D) two-color fluorescence dot plots for measuring backgroundfluorescence in high intensity areas for each channel (*-outlined forfluorescent gating) shows 0.149% fluorescence (*-upper left quadrant)and 0.00% (*-lower right quadrant); FIG. 62E) Cells fluorescentlystained with Epithelial Cell Adhesion Molecule (EpCAM) antibody (foridentifying epidermal cells), then gated for size as in A into atwo-color fluorescence dot plot, shows 83.4% EpCAM+ epithelial cells(*-outlined for fluorescent gating in upper left quadrant); and FIG.62F) Cells fluorescently stained with Vimentin, a type III intermediatefilament (IF) protein expressed in non-epithelial cells, then gated forsize as in A into a two-color fluorescence dot plot shows 15.6%Vimentin+non-epithelial cells (*-outlined for fluorescent gating inlower right quadrant).

FIG. 63A-D: Shows exemplary flow cytometry fluorescent dot plots of sizegated populations of enteroid iPS-derived intestinal cells that are notepithelial cells, from a microfluidic chip after 12 days of incubation.Cells were fluorescently stained with an antibody for identifying thefollowing cells as a percentage of the population gated intotwo-fluorescence plots: FIG. 63A) Paneth cells 5.03% (*-outlined in thelower right quadrant); FIG. 63B) Enteroendocrine cells 0.153%(*-outlined/fluorescently gated in the lower right quadrant); FIG. 63C)Goblet cells 0.131% (*-outlined/fluorescently gated in the lower rightquadrant); and FIG. 63D) Enterocytes 1.06% (*-outlined/fluorescentlygated in the lower right quadrant).

FIG. 64A-D: Shows exemplary flow cytometry fluorescent dot plots ofenteroid iPS-derived intestinal cells as percentages of epithelial andnonepithelial size gated cells from a microfluidic chip after 12 days ofincubation. Intestinal cell populations from size gated cells then gatedinto fluorescent intensity dot plots: FIG. 64A) Cells incubated with anisotype antibody control for the EpCAM primary antibody showing cellshaving 0.855% background fluorescence (*-outlined/gated in the upperleft quadrant); FIG. 64B) Cells incubated with secondary antibodywithout primary antibody having 0.065% background fluorescence(*-outlined/gated in the lower right quadrant); FIG. 64C)EpCAM+epithelial cells as 72% of the intestinal cell population; andFIG. 64D) Vimentin+non-epithelial cells: 28.6% of the intestinal cellpopulation.

EXAMPLE 43 Dividing Cells are Located in the Base of the IntestinalVilli—Pulse Chase Experiments

This example demonstrates that dividing cells are primarily located atthe base of the intestinal villi in the microfluidic intestinalorgan-on-chip.

As an example, general pulse-chase experiments for detecting DNA individing cells, cells are incubated with a labeling compound capable ofbeing incorporated into DNA as it is being replicated. As examples oflabeling compounds, certain thymidine (typically radioactive) orthymidine analogs (either containing a label or capable of being thetarget of a label), are used as labels incorporated into newlysynthesized DNA in mitotically active cells in the S-phase, the pulsecomponent. At chosen time-points, the labeling compound is washed out ofthe media and replaced with nonlabeled compounds with various times ofculture incubation to follow the fate of the cells, in some cases,following migration of and/or location of cells within a tissue.

While several radioactive and nonradioactive methods are used to detectand/or follow the label inside the nucleous of the dividing cells, themethod used herein incorporated a thymidine analog EdU(5-ethynyl-2′-deoxyuridine). Incorporation of EdU is detected throughits reaction with an azide dye that is small enough to penetrate tissuesefficiently. Visualization of EdU is rapid and typically does notinterfere with subsequent antibody staining.

Thus in one embodiment, EdU was pulsed for either 2 or 4 hrs. After thistime period, the EdU was removed (by washing out the media containingthe label) meaning that no more dividing cells could incorporate it intheir DNA and the chase component of the experiment now began. Thus insome embodiments, the chase incubation time was 24, 72 or 120 hours,i.e. an amount of time that the cells were cultured after the initialpulse of EdU.

In the figures shown herein, the vast majority of the dividing cells arelocated at the base after the pulse component of the experiment. Duringthe chase component of the experiment, at different time-points, theselabeled cells are found in upper parts of villi structures, thus thesebasal cells then travel up the sides and towards the tops of the villi.

FIG. 65A-C: Shows exemplary florescent micrographs of pulse-chasedmitotic/dividing cells in intestinal villi in a microfluidic chip. EdUlabeled (green) mitotic/dividing cells are shown in contrast toepithelial cells expressing E-cadherin (red) and nuclei stained withDAPI (blue). FIG. 65A) After a 4 hour pulse; then labeled cells areshown after FIG. 65B) a 72 hour chase and FIG. 65C) a 120 hour chase.

FIG. 66A-C: Shows exemplary florescent micrographs of pulse-chaseddividing cells located at the base of intestinal villi then moving intoupper villi structures growing in a microfluidic chip. EdU labeled(green) mitotic/dividing cells are shown in contrast to nuclei stainedwith DAPI (blue). EdU labeled (green) mitotic/dividing cells are locatedat the base of the intestinal microvilli FIG. 66A) after a 2 hour pulse;then labeled cells are located in villi structures after FIG. 66B) a 24hour chase and FIG. 66C) a 72 hour chase.

FIG. 67A-C: Shows exemplary florescent micrographs of pulse-chasedmitotic/dividing cells in intestinal villi in a microfluidic chip. EdUlabeled (green) mitotic/dividing cells are shown in contrast toepithelial cells expressing E-cadherin (red) and nuclei stained withDAPI (blue). EdU labeled (green) mitotic/dividing cells are located atthe base of the intestinal microvilli FIG. 67A) after a 2 hour pulse;then labeled cells are located in villi structures after FIG. 67B) a 24hour chase and FIG. 67C) a 72 hour chase.

FIG. 68A-C: Shows exemplary florescent micrographs of EdU labeledpulse-chased mitotic/dividing cells in intestinal villi in amicrofluidic chip as shown in FIG. 61. EdU labeled (green)mitotic/dividing cells are more clearly shown at the base of theintestinal microvilli without epithelial or nuclear stains FIG. 68A)after a 2 hour pulse; then labeled cells are located in villi structuresafter FIG. 68B) a 24 hour chase and FIG. 68C) a 72 hour chase.

EXAMPLE 44 Freezing iPS Cells for use in Multiple Experiments Over Time

One restriction on the use of intestinal enteroid cells derived fromhuman iPS cell lines is that these cells need to be used during acertain time period for producing viable and reproducible microfluidicintestinal chips. However, during the development of the presentinventions, methods and conditions were developed for using multiplealiquots (i.e. duplicate samples) of the same human intestinal enteroidcells in experiments separated by long time periods from the firstexperiment using these cells. Alternatively, intestinal enteroid cellsderived from human iPS cell lines may be stored long term before use ina microfluidic chip.

As an exemplary direct use method, iPS cells (i.e. human iPSC) arecultured for 36-37 days then undergo differentiation into intestinalorganoid cells over days 27-28. Oraganoid cells are then dissociatedinto single cell suspensions then a sub-population is selected forseeding microfluidic chips. The type of selection includes flow sorting,e.g. for EpCAM+ cells either by FACS or MACS, or selection may insteadbe done by the use of a selection reagent added to the organoid cellculture for detaching desired cells into a single cell suspention asdescribed herein. For reference, Magnetic-activated cell sorting (MACS)refers to a method for separation of various cell populations dependingon their surface molecules.

Regardless of the selection method used for providing a single cellsuspension, these single cells suspensions are directly used for seedingan apical channel of a microfluidic chip. After 7-14 days of cultureunder flow conditions, chips have epithelium containing villi asdescribed herein, see, FIG. 69A.

As an exemplary freezing method, iPS cells are cultured anddifferentiated into intestinal organoid cells then selected as describedabove. After the cells are selected for the desired subpopulation ofcells, they are re-suspended in Cryostor in a sterial cryogenicvial/tube. Cryostor refers to a defined cryopreservation medium, asexamples, CryoStor® CS10 (serum-free, animal component-free, and definedcryopreservation medium containing 10% dimethyl sulfoxide (DMSO), 5%DMASO in CryoStor® CS5 or 2% DMSO in CryoStor®CS2, obtained from StemCell Technolgies. Cryogenic vials containing intestinal iPS cells arethen frozen and stored in a liquid nitrogen tank. Upon thawing,previously frozen intestinal organoid cells were used for seeding chipsresulting in the same time frame of 7-14 days for producing epitheliumcontaining villi, see FIG. 69B. As an exemplary result, 66% survival(i.e. live cells) was observed upon thawing. Further, these thawed cellswere also placed (seeded) in trans-wells producing viable cultures thatgrew well.

FIG. 69A-B: Shows schematic diagrams of time line comparisons betweenintestinal enteroid cells derived from iPS cells. In one embodiment,cells are used FIG. 69A) directly or FIG. 69B) after freezing andthawing. Under both conditions, chips have epithelium containing villi(villous) structures.

EXAMPLE 45 Variations of Organ-Chip Designs

Additional embodiments of microfluidic organ-chip designs are shown inFIGS. 65-67, wherein micofludic chips for multiple organs are fludicallyattached

FIG. 70: Shows a schematic diagram of a 3 organ circuit, wherein 3micofludic chips for 3 different organ-on-chips are fluidically attachedthrough basal channels.

FIG. 71: Shows a schematic diagram of a 3 organ circuit, wherein 3micofludic chips for 3 different organ-on-chips are partiallyfluidically attached, i.e. through apical or basal channels.

FIG. 72: Shows a schematic diagram of a 2 organ circuit, wherein 2micofludic chips for 2 different organ-on-chips are partiallyfluidically attached, i.e. through the apical channels.

For reference, the upper-apical channed is shown in a solid green linewhile the lower-basasl channel is shown in a dotted red line.

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are sources of lymphoblastoidcells, pluripotent stem cells derived from therein, techniques andcomposition related to deriving pluripotent stem cells fromlymphoblastoid cells, differentiating techniques and compositions,biomarkers associated with such cells, and the particular use of theproducts created through the teachings of the invention. Variousembodiments of the invention can specifically include or exclude any ofthese variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

It is to be understood that the embodiments of the invention disclosedherein are illustrative of the principles of the present invention.Other modifications that can be employed can be within the scope of theinvention. Thus, by way of example, but not of limitation, alternativeconfigurations of the present invention can be utilized in accordancewith the teachings herein. Accordingly, embodiments of the presentinvention are not limited to that precisely as shown and described

It is to be understood that the embodiments of the invention disclosedherein are illustrative of the principles of the present invention.Other modifications that can be employed can be within the scope of theinvention. Thus, by way of example, but not of limitation, alternativeconfigurations of the present invention can be utilized in accordancewith the teachings herein. Accordingly, embodiments of the presentinvention are not limited to that precisely as shown and described.

1-38. (canceled)
 39. A method of culturing cells, comprising: a)providing i) iPS-derived organoids and ii) a fluidic device comprising amembrane, said membrane comprising a top surface and a bottom surface;b) disaggregating said iPS-derived organoids into single cells; c)seeding said single cells on said top or bottom surface of saidmembrane; and d) culturing said seeded cells under flow conditions thatsupport the maturation and/or differentiation of said seeded cells intointestinal cells.
 40. The method of claim 39, wherein said intestinalcells are selected from the group consisting of foregut intestinalepithelial cells, midgut intestinal epithelial cells and hindgutintestinal epithelial cells.
 41. The method of claim 39, wherein saidseeded cells differentiate into Paneth cells, endocrine cells and/orgoblet cells.
 42. The method of claim 39, wherein the iPS-derivedorganoid single cells are seeded on said top surface and cells of asecond type are seeded on said bottom surface.
 43. The method of claim39, wherein, the culturing under flow conditions results in theformation of villi.